Cationic lipid compositions for tissue-specific delivery

ABSTRACT

Provided herein are, inter alia, compositions and methods useful for the in vivo delivery of bioactive agents (e.g., therapeutic or diagnostic agents). The compositions provided herein include cationic lipids, helper lipids and a biostability enhancing agent, which together form a lipid aggregate with the bioactive agent and allow for the systemic delivery of the bioactive agent to, for example, lung tissue without the requirement for biomolecular targeting.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/552,783, filed Aug. 31, 2017, which disclosure isherein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Delivery of payloads such as therapeutic nucleic acids to specifictissues has traditionally been achieved using biomolecular targeting vialigand or receptor expression on the surface of lipid nanoparticleswhich serve as a delivery vehicle. Designing lipid nanoparticles capableof targeting specific organs, tissues, or cell types without the use ofcanonical biomolecular targeting techniques has been a significantchallenge. Manipulation of the inherent properties of lipidnanoparticles affords a means of achieving organ, tissue, and cell-typespecific targeting. Provided herein are compositions and methods whichcure this and other needs in the art.

BRIEF SUMMARY OF THE INVENTION

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio from about 0.18 to about 0.32 andof formula:

wherein R¹ and R² are independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R³and R⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; m isan integer from 1 to 6; X_(a) ⁻ is an anion; (ii) a second cationiclipid at a compositional molar ratio from about 0.24 to about 0.51 andof formula:

wherein R⁵ and R⁸ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁶ and R⁷ are independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 1 to 6; and X_(b) ⁻ is ananion; (iii) a first helper lipid; (iv) a second helper lipid; and (v) abiostability enhancing agent.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio from about 0.18 to about 0.32 andof formula:

wherein R¹ and R² are independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R³and R⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; m isan integer from 1 to 6; X_(a) ⁻ is an anion; (ii) a second cationiclipid at a compositional molar ratio from about 0.24 to about 0.51 andof formula:

wherein R⁵ and R⁸ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁶ and R⁷ are independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 1 to 6; and X_(b) ⁻ is ananion; (iii) a first helper lipid at a compositional molar ratio fromabout 0.20 to about 0.32; (iv) a second helper lipid; and (v) abiostability enhancing agent.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio from about 0.18 to about 0.32 andof formula:

wherein R¹ and R² are independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R³and R⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; m isan integer from 1 to 6; X_(a) ⁻ is an anion; (ii) a second cationiclipid at a compositional molar ratio from about 0.24 to about 0.51 andof formula:

wherein R⁵ and R⁸ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁶ and R⁷ are independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 1 to 6; and X_(b) ⁻ is ananion; (iii) a first helper lipid at a compositional molar ratio fromabout 0.20 to about 0.32; (iv) a second helper lipid at a compositionalmolar ratio from about 0.01 to about 0.14; and (v) a biostabilityenhancing agent.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio from about 0.18 to about 0.32 andof formula:

wherein R¹ and R² are independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R³and R⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; m isan integer from 1 to 6; X_(a) ⁻ is an anion; (ii) a second cationiclipid at a compositional molar ratio from about 0.24 to about 0.51 andof formula:

wherein R⁵ and R⁸ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁶ and R⁷ are independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 1 to 6; and X_(b) ⁻ is ananion; (iii) a first helper lipid at a compositional molar ratio fromabout 0.20 to about 0.32; (iv) a second helper lipid at a compositionalmolar ratio from about 0.01 to about 0.14; and (v) a biostabilityenhancing agent at a compositional molar ratio from about 0.01 to about0.02.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.24, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.05, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.01, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.32, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.39, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.26, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.01, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.18, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.23, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.45, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.20, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.18, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.51, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.20, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.01, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.27, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.01, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.25, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.26, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.01, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.28, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.24, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.14, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 750.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.18, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.47, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.01, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein saidbiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.24, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.40, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.24, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 2000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.32, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.20, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.08, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a pharmaceutical composition including acomposition as provided herein including embodiments thereof and apharmaceutically acceptable excipient.

In an aspect is provided a cell including a composition as providedherein including embodiments thereof. In embodiments, the cell is amammalian cell. In embodiments, the cell is a rodent cell. Inembodiments, the cell is a mouse cell. In embodiments, the cell is a ratcell. In embodiments, the cell is a porcine cell. In embodiments, thecell is a canine cell. In embodiments, the cell is a primate cell. Inembodiments, the cell is an epithelial cell. In embodiments, the cell isan epithelial lung cell. In embodiments, the cell is an endothelialcell. In embodiments, the cell is an endothelial lung cell.

In an aspect is provided, method of delivering a bioactive agent to acell, the method including: (i) admixing an bioactive agent with acomposition as provided herein including embodiments thereof, therebyforming a bioactive agent-lipid complex; (ii) contacting a cell with thebioactive agent-lipid complex, thereby delivering the bioactiveagent-lipid complex to a cell.

In another aspect is provided a method of delivering a bioactive agentto lung tissue in a subject, the method including: (i) admixing anbioactive agent with a composition as described herein includingembodiments thereof, thereby forming a bioactive agent-lipid complex;(ii) systemically administering an effective amount of the bioactiveagent-lipid complex to a subject, thereby delivering the bioactiveagent-lipid complex to a lung tissue in a subject.

In another aspect is provided a method of expressing a protein in lungtissue in a subject, the method including: (i) admixing a mRNA with acomposition as described herein including embodiments thereof, therebyforming a mRNA-lipid complex; (ii) administering an effective amount ofthe mRNA-lipid complex to a subject; and (iii) allowing the mRNA of themRNA-lipid complex to express in lung tissue of the subject, therebyexpressing a protein in lung tissue in a subject.

In an aspect is provided a method of treating a pulmonary disease in asubject in need thereof, the method including administering to a subjecta therapeutically effective amount of a bioactive agent and acomposition as described herein including embodiments thereof, therebytreating a pulmonary disease in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-IC. Based on mixture Design of Experiment optimization, new LNPformulations were made, complexed with mRNA using the previouslydeveloped protocol (FIGS. 1A-1C), and the subsequent LNPs were screenedfor delivery in vivo using a luciferase readout. Top performingformulations were then used to model and predict second generationformulations for optimized tissue specific expression. These newformulations were then tested in vivo to identify the best LNPformulation. FIG. 1A. Cartoon representation of method for generatingnew lipid nanoparticle (LNP) formulations, testing in vivo, andoptimizing. FIG. 1B. Cartoon representation of steps involved incomplexing the LNP formulation with mRNA and testing in vivo. FIG. 1C.Written procedure of steps involved in complexing the LNP formulationwith mRNA and testing in vivo.

FIG. 2. In vivo luciferase assay workflow for LNP testing.

FIGS. 3A-3E. Systemic delivery of LNPs results in varied biodistributionpatterns. Whole animal in vivo imaging revealed organ specificbiodistribution patterns. FIG. 3A shows through whole animal imagingthat LNPs distributed in the spleen. FIG. 3B shows through whole animalimaging that LNPs distributed in the lung. FIGS. 3C-3E showbiodistribution patterns of LNPs determined from imaging ex vivoisolated organ tissue.

FIGS. 4A-4B. Performance optimization screening. FIG. 4A. Lung directeddelivery: ex vivo quantification. FIG. 4B. Spleen directed delivery: exvivo quantification.

FIGS. 5A-5C. Dose dependent protein expression. FIG. 5A. Lung deliveryin vivo luciferase expression. FIG. 5B. Lung radiance as a function ofmRNA dose titration (3 mg/kg, 1 mg/kg, 0.5 mg/kg) quantified 4 hours (4h) post-injection. FIG. 5C. Lung directed delivery: in vivoquantification. In vivo radiance over time (hours) quantified for eachdose titration (3 mg/kg, 1 mg/kg, 0.5 mg/kg).

FIGS. 6A-6C. Time course of protein expression. FIG. 6A. Ex vivo lungbioluminescence signal. FIG. 6B. In vivo bioluminescence signal. FIG.6C. Quantification of ex vivo lung luciferase signal.

FIGS. 7A-7J. Toxicity—Cytokine panel. Quantification of circulatingcytokine levels in mouse serum at 2 hrs, 4 hrs, 24 hrs and 48 hrspost-injection under two mRNA dose conditions: low (1 mg/kg) and high (3mg/kg). FIG. 7A. Levels of IL5. FIG. 7B. Levels of IL4. FIG. 7C. Levelsof IL2. FIG. 7D. Levels of IL10. FIG. 7E. Levels of IL1b. FIG. 7F.Levels of IL6. FIG. 7G. Levels of IFNg. FIG. 7H. Levels of GMCSF. FIG.7I. Levels of IL12. FIG. 7J. Levels of TNFa.

FIGS. 8A-8B. N/P ratio variance allows for tailoring of mRNA deliveryand expression, resulting in exclusive tissue expression patterns. Invivo whole animal and ex vivo isolated organ imaging indicates varyingN/P ratio can lead to exclusion of protein expression from certainorgans resulting in specific expression in the lung (FIG. 8A) or spleen(FIG. 8B).

FIG. 9. Lung delivery quantification assay: Luciferase assay.

FIGS. 10A-10B. Low performance formulations. Luciferase activity of lowperformance formulations with (FIG. 10B) and without (FIG. 10A) the bestformulation as a benchmark to compare.

FIGS. 11A-11F. Jet PEI® vs Bruce #3.14. In vivo whole animal imagingshows Bruce #3.14 lipid nanoparticle formulation has specificbiodistribution patterns (FIG. 11A) compared with Jet PEI® (FIG. 11B).Ex vivo isolated organ tissue imaging also demonstrates that Bruce #3.14lipid nanoparticle formulation has specific biodistribution patterns(FIG. 11C) compared with Jet PEI® (FIG. 11D). FIG. 11E shows Bruce #3.14lipid nanoparticle formulation and Jet PEI® flux. Two animals in the JetPEI® group died. FIG. 11F shows flux in lung after treatment with Bruce#3.14 lipid nanoparticle formulation or Jet PEI®.

FIGS. 12A-12C. Formulation #3.10 treatment. FIG. 12A shows organ fluxluciferase activity across organs. FIG. 12B shows in vivo whole animalluminescence. FIG. 12C shows ex vivo isolated organ tissue luminescence.

FIGS. 13A-13C. Formulation #3.14 treatment. FIG. 13A shows organ fluxluciferase activity across organs. FIG. 13B shows in vivo whole animalluminescence. FIG. 13C shows ex vivo isolated organ tissue luminescence.

FIGS. 14A-14C. Formulation #3.19 treatment. FIG. 14A shows organ fluxluciferase activity across organs. FIG. 14B shows in vivo whole animalluminescence. FIG. 14C shows ex vivo isolated organ tissue luminescence.

FIGS. 15A-15C. Formulation #3.20 treatment. FIG. 15A shows organ fluxluciferase activity across organs. FIG. 15B shows in vivo whole animalluminescence. FIG. 15C shows ex vivo isolated organ tissue luminescence.

FIGS. 16A-16C. Formulation #2.2 treatment. FIG. 16A shows organ fluxluciferase activity across organs. FIG. 16B shows in vivo whole animalluminescence. FIG. 16C shows ex vivo isolated organ tissue luminescence.

FIGS. 17A-17K. Bruce #3.14 in vivo/ex vivo time course (3 mg/kg). Invivo whole animal luminescence at 4 hrs (FIG. 17A), 24 hrs (FIG. 17B),48 hrs (FIG. 17C), 72 hrs (FIG. 17D), and 96 hrs (FIG. 17E). Ex vivoisolated organ tissue luminescence at 4 hrs (FIG. 17F), 24 hrs (FIG.17G), 48 hrs (FIG. 17H), 72 hrs (FIG. 17I), and 96 hrs (FIG. 17J). FIG.17K shows mean (total flux (p/s)) vs. column 1 over time.

FIGS. 18A-18C. mRNA dose variation and time course 3.14 (3, 1, 0.5mg/kg). In vivo whole animal luminescence at 4 hrs (FIG. 18A), 24 hrs(FIG. 18B), and 48 hrs (FIG. 18C) for each dose variation.

FIG. 19. Diagram of workflow.

FIGS. 20A-20B. Reagent comparative analysis. Experimentaldesign—Reagents: Four different formulations (IVF® Lung, DOTMA:DOPE,DOTAP:DOPE, Jet PEI®), Route of Delivery: Intravenous (systemic),Payload: Trilink® Firefly luciferase mRNA. FIG. 20A shows a bar graphdepicting lung radiance for each formulation. FIG. 20B shows in vivowhole animal radiance for each formulation.

FIG. 21. In vivo screening of different mRNA's encoding for FireflyLuciferase. mRNA expression kinetics can differ based on optimization ofmRNA. Experimental design—Reagent: Invivofectamine® Lung, Route ofDelivery: Intravenous (systemic), Payload: 2 different mRNAs encodingfor Firefly Luciferase (Trilink® and In House). Graph shows lungradiance for each payload used.

FIG. 22. siRNA knockdown in the lung. Tyrosine kinase receptor Tie2,also known as Tek, plays an important role in embryonic vasculature andpersists in adult endothelial cells. It is expressed almost exclusivelyin endothelial cells. Knockdown analysis was performed on isolated lungtissue 48 hours post IV delivery of LNPs (IVF Rx lung+siRNA) (N=4).Graph shows the percentage of Tie2 remaining.

FIG. 23. Cartoon representation of lacZ Cre-reporter strain and theresult of delivering Cre mRNA using IVF® Lung.

FIGS. 24A-24D. lacZ mRNA delivery in wild type mice. FIGS. 24A and 24Cshow lacZ expression via beta-gal staining. FIGS. 24B and 24D are highmagnification images of the regions circled in 24A and 24C,respectively.

FIGS. 25A-25B. Cre mRNA delivery in lacZ reporter mice. Invivofectamine®Lung delivery in vivo—Immunofluorescent staining for detection of lacZexpression reveals expression throughout the lung tissue, and isdistinctly visible in bronchiole structures. FIG. 25A shows 10× view ofstaining. FIG. 25B shows 25× view of staining.

FIG. 26. Cartoon illustration describing the N/P ratio. N/P ratio refersto the positive charge contributed by Nitrogen residues on the cationiclipid versus the negative charge contributed by the Phosphates on thenucleic acid backbone. High N/P=Greater amounts of lipid compared tomRNA. Low N/P=Lower amounts of lipid compared to mRNA. N/P ratio greatlyaffects the surface charge of the lipid nanoparticle which stronglygoverns transfection efficiency and affects biodistribution.

FIGS. 27A-27K. N/P ratio affects biodistribution pattern. In vivo wholeanimal imaging and ex vivo isolated organ tissue imaging showing theeffect of N/P ratio (10, 8, 6, 4, 2) on biodistribution. FIGS. 27A and27B shows N/P ratio 10. FIGS. 27C and 27D shows N/P ratio 8. FIGS. 27Eand 27F shows N/P ratio 6. FIGS. 27G and 27H shows N/P ratio 4. FIGS.27I and 27J shows N/P ratio 2. FIG. 27K shows a side view of N/P ratio4.

FIG. 28. Formulation: DHDMS/DOPE with N/P ratio alteration. No lungdelivery seen and varied N/P ratio did not affect lung distribution. Exvivo isolated organ tissue images.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedchain, or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e., C₁-C₁₀ means one to tencarbons). Alkyl is an uncyclized chain. Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example,n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkylgroup is one having one or more double bonds or triple bonds. Examplesof unsaturated alkyl groups include, but are not limited to, vinyl,2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. An alkoxy is an alkyl attached to theremainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred. A “lower alkyl” or“lower alkylene” is a C₁-C₈ alkyl or alkylene group.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, consisting of at least one carbon atom and atleast one heteroatom selected from the group consisting of O, N, P, Si,and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized, and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) 0, N, P, S, and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Heteroalkyl is an uncyclizedchain. Examples include, but are not limited

to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheteroalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain from one to four heteroatoms selected from N, O, and S,wherein the nitrogen and sulfur atoms are optionally oxidized, and thenitrogen atom(s) are optionally quaternized. Thus, the term “heteroaryl”includes fused ring heteroaryl groups (i.e., multiple rings fusedtogether wherein at least one of the fused rings is a heteroaromaticring). A 5,6-fused ring heteroarylene refers to two rings fusedtogether, wherein one ring has 5 members and the other ring has 6members, and wherein at least one ring is a heteroaryl ring. Likewise, a6,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 6 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 5 members, and wherein at least one ring is aheteroaryl ring. A heteroaryl group can be attached to the remainder ofthe molecule through a carbon or heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl, and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylsulfonyl,” as used herein, means a moiety having theformula —S(O₂)—R′, where R′ is an alkyl group as defined above. R′ mayhave a specified number of carbons (e.g., “C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″, and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compounddisclosed herein includes more than one R group, for example, each ofthe R groups is independently selected as are each R′, R″, R′″, and R″″group when more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number ranging fromzero to the total number of open valences on the aromatic ring system;and where R′, R″, R′″, and R″″ are preferably independently selectedfrom hydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, and substituted or unsubstituted heteroaryl. When acompound disclosed herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ groups when more than one of these groups is present.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In embodiments, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from: oxo, —OH, —NH₂, —SH, —CN,                —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth herein.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls. Moreover, where a moiety is substitutedwith an R substituent, the group may be referred to as “R-substituted.”Where a moiety is R-substituted, the moiety is substituted with at leastone R substituent and each R substituent is optionally different.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single-, double- or multiple-stranded form,or complements thereof. The term “polynucleotide” refers to a linearsequence of nucleotides. The term “nucleotide” typically refers to asingle unit of a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA andmRNA), and hybrid molecules having mixtures of single and doublestranded DNA and RNA. Nucleic acids can be linear or branched. Forexample, nucleic acids can be a linear chain of nucleotides or thenucleic acids can be branched, e.g., such that the nucleic acidscomprise one or more arms or branches of nucleotides. Optionally, thebranched nucleic acids are repetitively branched to form higher orderedstructures such as dendrimers and the like.

Nucleic acids, including nucleic acids with a phosphothioate backbonecan include one or more reactive moieties. As used herein, the termreactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The terms also encompass nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphodiester derivativesincluding, e.g., phosphoramidate, phosphorodiamidate, phosphorothioate(also known as phosphothioate), phosphorodithioate, phosphonocarboxylicacids, phosphonocarboxylates, phosphonoacetic acid, phosphonoformicacid, methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, Oligonucleotides and Analogues: A PracticalApproach, Oxford University Press); and peptide nucleic acid backbonesand linkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g. phosphorodiamidate morpholino oligos or locked nucleicacids (LNA)), including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. In embodiments, the internucleotide linkages in DNAare phosphodiester, phosphodiester derivatives, or a combination ofboth.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may be conjugated to a moiety that does not consistof amino acids. The terms apply to amino acid polymers in which one ormore amino acid residue is an artificial chemical mimetic of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymers. The terms apply to macrocyclic peptides, peptides that havebeen modified with non-peptide functionality, peptidomimetics,polyamides, and macrolactams. A “fusion protein” refers to a chimericprotein encoding two or more separate protein sequences that arerecombinantly expressed as a single moiety.

The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, in general the amino acid residue number in a test sequencedetermined by simply counting from the N-terminus will not necessarilybe the same as the number of its corresponding position in the referencesequence. For example, in a case where a variant has a deletion relativeto an aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned referencesequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The terms “numbered with reference to” or “corresponding to,” when usedin the context of the numbering of a given amino acid or polynucleotidesequence, refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,or 99% identity over a specified region, e.g., of the entire polypeptidesequences of the invention or individual domains of the polypeptides ofthe invention), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. Such sequences are then said to be “substantiallyidentical.” This definition also refers to the complement of a testsequence. Optionally, the identity exists over a region that is at leastabout 50 nucleotides in length, or more preferably over a region that is100 to 500 or 1000 or more nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of, e.g., a full length sequence or from 20 to 600, about 50to about 200, or about 100 to about 150 amino acids or nucleotides inwhich a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross-reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated, however, that the resulting reaction product can beproduced directly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture. In embodiments contacting includes,for example, allowing a ribonucleic acid as described herein to interactwith a an endonuclease and an enhancer element.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Acontrol can also represent an average value gathered from a number oftests or results. One of skill in the art will recognize that controlscan be designed for assessment of any number of parameters. For example,a control can be devised to compare therapeutic benefit based onpharmacological data (e.g., half-life) or therapeutic measures (e.g.,comparison of side effects). One of skill in the art will understandwhich standard controls are most appropriate in a given situation and beable to analyze data based on comparisons to standard control values.Standard controls are also valuable for determining the significance(e.g. statistical significance) of data. For example, if values for agiven parameter are widely variant in standard controls, variation intest samples will not be considered as significant.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include ³²P,fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget peptide. Any appropriate method known in the art for conjugatingan antibody to the label may be employed, e.g., using methods describedin Hermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego.

A “labeled protein or polypeptide” is one that is bound, eithercovalently, through a linker or a chemical bond, or noncovalently,through ionic, van der Waals, electrostatic, or hydrogen bonds to alabel such that the presence of the labeled protein or polypeptide maybe detected by detecting the presence of the label bound to the labeledprotein or polypeptide. Alternatively, methods using high affinityinteractions may achieve the same results where one of a pair of bindingpartners binds to the other, e.g., biotin, streptavidin.

“Biological sample” or “sample” refer to materials obtained from orderived from a subject or patient. A biological sample includes sectionsof tissues such as biopsy and autopsy samples, and frozen sections takenfor histological purposes. Such samples include bodily fluids such asblood and blood fractions or products (e.g., serum, plasma, platelets,red blood cells, and the like), sputum, tissue, cultured cells (e.g.,primary cultures, explants, and transformed cells) stool, urine,synovial fluid, joint tissue, synovial tissue, synoviocytes,fibroblast-like synoviocytes, macrophage-like synoviocytes, immunecells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Abiological sample is typically obtained from a eukaryotic organism, suchas a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat;a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; orfish.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

The word “expression” or “expressed” as used herein in reference to agene means the transcriptional and/or translational product of thatgene. The level of expression of a DNA molecule in a cell may bedetermined on the basis of either the amount of corresponding mRNA thatis present within the cell or the amount of protein encoded by that DNAproduced by the cell (Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, 18.1-18.88).

Expression of a transfected gene can occur transiently or stably in acell. During “transient expression” the transfected gene is nottransferred to the daughter cell during cell division. Since itsexpression is restricted to the transfected cell, expression of the geneis lost over time. In contrast, stable expression of a transfected genecan occur when the gene is co-transfected with another gene that confersa selection advantage to the transfected cell. Such a selectionadvantage may be a resistance towards a certain toxin that is presentedto the cell.

The term “exogenous” refers to a molecule or substance (e.g., nucleicacid or protein) that originates from outside a given cell or organism.Conversely, the term “endogenous” refers to a molecule or substance thatis native to, or originates within, a given cell or organism.

A “cell culture” is an in vitro population of cells residing outside ofan organism. The cell culture can be established from primary cellsisolated from a cell bank or animal, or secondary cells that are derivedfrom one of these sources and immortalized for long-term in vitrocultures.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

The compositions can be administered for therapeutic or prophylactictreatments. In therapeutic applications, compositions are administeredto a patient suffering from a disease (e.g., pulmonary disease) in a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. A “patient” or “subject” for the purposesof the present invention includes both humans and other animals,particularly mammals. Thus the methods are applicable to both humantherapy, veterinary applications, and in research use settings, forexample in experimental animal models including rodent, canine, andprimate animal models. In certain embodiments the subject or patient isa mammal, preferably a primate, and in the most preferred embodiment thepatient is human. In other embodiments the subject or patient is amammal, preferably a rodent, and in the most preferred embodiments amouse or rat.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

The compositions provided herein, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by intravenous infusion,intraperitoneally, intravesically or intrathecally. Parenteraladministration, and intravenous administration are the preferred methodsof administration. The formulations of compounds can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as powders in vials or ampoules. The composition can,if desired, also contain other compatible therapeutic agents.

The combined administrations contemplates co-administration, usingseparate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities.

Effective doses of the compositions provided herein vary depending uponmany different factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. However, a person of ordinary skill in theart would immediately recognize appropriate and/or equivalent doseslooking at dosages of approved compositions for treating and preventinglung/pulmonary disorders for guidance.

The terms “disease” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In embodiments,the disease is a pulmonary disease (e.g. lung cancer, asthma, chronicobstructive pulmonary disease (COPD), cystic fibrosis).

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g., lungdisease, lung cancer, asthma, chronic obstructive pulmonary disease(COPD), cystic fibrosis) is caused by (in whole or in part), or asymptom of the disease is caused by (in whole or in part) the substanceor substance activity or function.

The terms “transfection”, “transduction”, “transfecting” or“transducing” can be used interchangeably and are defined as a processof introducing a nucleic acid molecule and/or a protein and/or aribonucleoprotein to a cell in culture or in a tissue in vivo. Thenucleic acid molecule can be a sequence encoding complete proteins,ribonucleoproteins or functional portions thereof. Typically, a nucleicacid encoding proteins, ribonucleoproteins or functional portionsthereof comprises the elements necessary for expression of the proteinor functional portion thereof (e.g., a promoter, transcription startsite, etc.). Non-viral methods of transfection include any appropriatemethod that does not use viral DNA or viral particles as a deliverysystem to introduce the nucleic acid molecule into the cell. Exemplarynon-viral transfection methods include liposomal transfection. The terms“transfection” or “transduction” also refer to introducing nucleic acidsand/or proteins into a cell from the external environment. Throughtransfection, the nucleic acid molecule and/or a protein and/or aribonucleoprotein is delivered into the interior of the cell or thecells constituting the tissue. Transfection of a nucleic acid moleculeand/or a protein and/or a ribonucleoprotein into a cell or tissue may beperformed with the purpose of modifying the biological function of thecell. Alternatively, transfection of a nucleic acid molecule and/or aprotein and/or a ribonucleoprotein may be performed with the purpose ofdelivering a detectable label to a cell or tissue to facilitateidentification of a cell or tissue. For example, nucleic acids (e.g.,DNA, RNA, mRNA, siRNA, miRNA, guide RNA) and/or ribonucleoproteins(e.g., CAS 9) can be introduced into a cell or tissue via lipid-mediateddelivery (e.g., liposomal transfection). The nucleic acid molecule mayalternatively be an mRNA, a siRNA, a miRNA or a guide RNA. In someinstances, the nucleic acid molecule may be bound to a ribonucleoprotein(e.g., Cas9 bound to a guide RNA).

For specific proteins described herein, the named protein includes anyof the protein's naturally occurring forms, or variants or homologs thatmaintain the protein transcription factor activity (e.g., within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity comparedto the native protein). In some embodiments, variants or homologs haveat least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring form. In other embodiments, the protein is theprotein as identified by its NCBI sequence reference. In otherembodiments, the protein is the protein as identified by its NCBIsequence reference or functional fragment or homolog thereof.

Thus, a “CRISPR associated protein 9,” “Cas9” or “Cas9 protein” asreferred to herein includes any of the recombinant ornaturally-occurring forms of the Cas9 endonuclease or variants orhomologs thereof that maintain Cas9 endonuclease enzyme activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to Cas9). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring Cas9 protein. In embodiments, the Cas9 protein issubstantially identical to the protein identified by the UniProtreference number Q99ZW2 or a variant or homolog having substantialidentity thereto. Cas9 refers to the protein also known in the art as“nickase”. In embodiments, Cas9 binds a CRISPR (clustered regularlyinterspaced short palindromic repeats) nucleic acid sequence. Inembodiments, the CRISPR nucleic acid sequence is a prokaryotic nucleicacid sequence.

As used herein, the term “lipid” refers to lipid molecules that caninclude fats, waxes, steroids, cholesterol, fat-soluble vitamins,monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids,cationic or anionic lipids, derivatized lipids, and the like, asdescribed in detail below.

Suitable phospholipids include but are not limited tophosphatidylcholine (PC), phosphatidic acid (PA),phosphatidylethanolamine (PE), phosphatidylglycerol (PG),phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristoylphosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC),dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline(DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoylphosphatidyl glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG),dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidylserine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoylphosphatidyl serine (DOPS), dipalmitoyl phosphatidyl serine (DPPS),dioleoyl phosphatidyl ethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE),1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE), andcardiolipin. In some embodiments, the phospholipid is DOPE. In otherembodiments, the phospholipid is DSPC. Lipid extracts, such as egg PC,heart extract, brain extract, liver extract, and soy PC, are also usefulin the present invention. In some embodiments, soy PC can include HydroSoy PC (HSPC). In certain embodiments, the lipids can includederivatized lipids, such as PEGylated lipids. Derivatized lipids caninclude, for example, DSPE-PEG2000, cholesterol-PEG2000,DSPE-polyglycerol, or other derivatives generally known in the art.

A “cationic lipid” as provided herein refers, in the usual and customarysense, to a net positively charged lipid which can facilitate theformation of lipid aggregates. Cationic lipids contain positivelycharged functional groups under physiological conditions. Cationiclipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammoniumbromide (DMRIE), N-[1-(2,3,dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), 3β-[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB) andN,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA). In embodiments, thecationic lipid (e.g., first cationic lipid, second cationic lipid) is acompound of formula (I) or (II). In embodiments, the first cationiclipid is dihydroxy dimyristyl spermidine (DHDMS). In embodiments, thesecond cationic lipid is hydroxy dimyristyl spermidine (HDMS).

Lipids can form micelles, monolayers, and bilayer membranes. The lipidscan self-assemble into liposomes or lipid aggregates.

The term “lipid aggregate” refers to a lipid structure including aplurality of lipids or type of lipids, forming a higher order structure(e.g., secondary, tertiary or quaternary structure). Non-limitingexamples of lipid aggregates include liposomes, unilamellar vesicles,multilamellar vesicles, micelles, amorphous aggregates, and the like.The lipid aggregates of the present invention can contain any suitablelipid, including cationic lipids, zwitterionic lipids, neutral lipids,or anionic lipids. In embodiments, the lipid aggregate includes acationic lipid or a cationic lipid type. In embodiments, the lipidaggregate includes a cationic lipid or a cationic lipid type incombination with a non-cationic (e.g., neutral) lipid or a non-cationiclipid type. In embodiments, the lipid aggregate has a net positivecharge. In embodiments, the lipid aggregate includes a cationic lipidand a neutral lipid. In embodiments, the cationic lipid is a cationiclipid as described in U.S. Pat. No. 8,785,200 which is herebyincorporated by reference and for all purposes.

In embodiments, the lipid aggregate includes a single lipid. Inembodiments, the lipid aggregate includes a plurality of differentlipids (e.g., first cationic lipid, second cationic lipid, helperlipid). Where the lipid aggregate includes a plurality of differentlipids the lipid aggregate may include a lipid blend. A “lipid blend” asprovided herein is a mixture of a plurality of lipid types. Inembodiments, the lipid blend includes a first lipid type, a second lipidtype or a third lipid type. The first, second and third lipid type maybe independently different (e.g., cationic lipid and non-cationiclipid). Therefore, a person having ordinary skill in the art willimmediately recognize that the terms “lipid” and “lipid type(s)” havethe same meaning and can be used interchangeably.

In embodiments, the lipid aggregate provided herein is a liposome. Asused herein, the term “liposome” encompasses any compartment enclosed bya lipid bilayer. The term liposome includes unilamellar vesicles whichare comprised of a single lipid bilayer and generally have a diameter inthe range of about 20 to about 400 nm. Liposomes can also bemultilamellar having a diameter in the range of approximately 1 μm toapproximately 10 μm. Multilamellar liposomes may consist of several(anywhere from two to hundreds) unilamellar vesicles forming one insidethe other in diminishing size, creating a multilamellar structure ofconcentric phospholipid spheres separated by layers of water.Alternatively, multilamellar liposomes may consist of many smaller nonconcentric spheres of lipid inside a large liposome. In embodiments,liposomes include multilamellar vesicles (MLV), large unilamellarvesicles (LUV), and small unilamellar vesicles (SUV). The liposomes ofthe present invention can contain any suitable lipid, including cationiclipids, zwitterionic lipids, neutral lipids, or anionic lipids.

Compositions

Provided herein are, inter alia, compositions and methods useful for thein vivo delivery of bioactive agents (e.g., therapeutic, biologicallyactive, or diagnostic agents). The compositions and methods providedherein including embodiments thereof may be, inter alia, used for thedelivery of bioactive agents (e.g., nucleic acid molecules,ribonucleoproteins, small molecules or combinations thereof) to the lung(e.g., including but not limited to endothelial lung cells, epitheliallung cells) of a subject. The compositions provided herein includecationic lipids, helper lipids and a biostability enhancing agent, whichtogether form a lipid aggregate with the bioactive agent and allow forthe systemic delivery of the bioactive agent to, for example, lungtissue without the requirement for biomolecular targeting.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio from about 0.18 to about 0.32 andof formula:

In formula (I) R¹ and R² are independently substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. R³ and R⁴ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. m is an integer from 1 to 6. X_(a) ⁻ is ananion. (ii) A second cationic lipid at a compositional molar ratio fromabout 0.24 to about 0.51 and of formula:

In formula (II) R⁵ and R⁸ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R⁶ and R⁷ are independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. n is an integer from 1 to 6. X_(b) ⁻ is ananion. (iii) A first helper lipid at a compositional molar ratio fromabout 0.20 to about 0.32. (iv) A second helper lipid at a compositionalmolar ratio from about 0.01 to about 0.14; and (v) a biostabilityenhancing agent at a compositional molar ratio from about 0.01 to about0.02.

In embodiments, R¹ and R² are independently substituted or unsubstitutedalkyl. In embodiments, R¹ and R² are independently unsubstituted alkyl.R¹ and R² are independently unsubstituted C₁-C₂₀ alkyl. In embodiments,R¹ and R² are independently unsubstituted C₅-C₂₀ alkyl. In embodiments,R¹ and R² are independently unsubstituted C₁₀-C₂₀ alkyl. In embodiments,R¹ and R² are independently unsubstituted C₁₂-C₁₈ alkyl. In embodiments,R¹ and R² are independently unsubstituted C₁₄-C₁₆ alkyl. In embodiments,R¹ is unsubstituted C₁₄ alkyl. In embodiments, R² is unsubstituted C₁₄alkyl. In embodiments, R¹ is unsubstituted C₁₅ alkyl. In embodiments, R²is unsubstituted C₁₅ alkyl. In embodiments, R¹ is unsubstituted C₁₆alkyl. In embodiments, R² is unsubstituted C₁₆ alkyl. In embodiments, R¹is —(CH₂)₁₃CH₃. In embodiments, R² is —(CH₂)₁₃CH₃.

In embodiments, R³ and R⁴ are independently hydrogen or substituted orunsubstituted alkyl. In embodiments, R³ and R⁴ are independentlyhydrogen.

In embodiments, R⁵, R⁶ and R⁷ are independently hydrogen, substituted orunsubstituted alkyl. In embodiments, R⁵, R⁶ and R⁷ are independentlyhydrogen or unsubstituted alkyl. In embodiments, R⁵, R⁶ and R⁷ areindependently hydrogen or unsubstituted C₁-C₂₀ alkyl. In embodiments,R⁵, R⁶ and R⁷ are independently hydrogen or unsubstituted C₅-C₂₀ alkyl.In embodiments, R⁵, R⁶ and R⁷ are independently hydrogen orunsubstituted C₁₀-C₂₀ alkyl. In embodiments, R⁵, R⁶ and R⁷ areindependently hydrogen or unsubstituted C₁₂-C₁₈ alkyl. In embodiments,R⁵, R⁶ and R⁷ are independently hydrogen or unsubstituted C₁₄-C₁₆ alkyl.In embodiments, R⁵, R⁶ and R⁷ are independently hydrogen orunsubstituted C₁₄ alkyl. In embodiments, R⁵, R⁶ and R⁷ are independentlyhydrogen or unsubstituted C₁₅ alkyl. In embodiments, R⁵, R⁶ and R⁷ areindependently hydrogen or unsubstituted C₁₆ alkyl. In embodiments, R⁵ isunsubstituted C₁₄ alkyl. In embodiments, R⁷ is unsubstituted C₁₄ alkyl.In embodiments, R⁵ is —(CH₂)₁₃CH₃. In embodiments, R⁶ is hydrogen. Inembodiments, R⁷ is —(CH₂)₁₃CH₃.

In embodiments, R⁸ is hydrogen or substituted or unsubstituted alkyl. Inembodiments, R⁸ is hydrogen.

In embodiments, m is an integer from about 1 to 6. In embodiments, m isan integer from about 1 to 5. In embodiments, m is an integer from about1 to 4. In embodiments, m is an integer from about 1 to 3. Inembodiments, m is an integer from 1 to 6. In embodiments, m is aninteger from 1 to 5. In embodiments, m is an integer from 1 to 4. Inembodiments, m is an integer from 1 to 3. In embodiments, m is 1. Inembodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. Inembodiments, m is 5. In embodiments, m is 6.

In embodiments, n is an integer from about 1 to 6. In embodiments, n isan integer from about 1 to 5. In embodiments, n is an integer from about1 to 4. In embodiments, n is an integer from about 1 to 3. Inembodiments, n is an integer from 1 to 6. In embodiments, n is aninteger from 1 to 5. In embodiments, n is an integer from 1 to 4. Inembodiments, n is an integer from 1 to 3. In embodiments, n is 1. Inembodiments, n is 2. In embodiments, n is 3. In embodiments, n is 4. Inembodiments, n is 5. In embodiments, n is 6.

In one embodiment, R¹ is —(CH₂)₁₃CH₃, R² is —(CH₂)₁₃CH₃, R³ is hydrogen,R⁴ is hydrogen, m is 4 and X_(a) ⁻ is CH₃COO⁻.

In one embodiment, R⁵ is —(CH₂)₁₃CH₃, R⁶ is hydrogen, R⁷ is —(CH₂)₁₃CH₃,R⁸ is hydrogen, n is 4 and X_(b) ⁻ is CH₃COO⁻.

The term “compositional molar ratio” of a compound (e.g., cationiclipid, helper lipid, biostability enhancing agent) refers to the ratioof the number of solute moles of an individual compound to the totalnumber of solute moles of all compounds in a solution. For example, thetotal number of solute moles may be 33.8 and the number of solute molesof an individual compound (e.g., first cationic lipid) may be 8.1resulting in a compositional ratio for the individual compound of 0.24.In embodiments, the total number of solute moles is 33.8 and the numberof solute moles of a single compound is 10.8 moles resulting in acompositional ratio of the single compound of 0.32.

The compositions provided herein include two or more helper lipids(e.g., a first helper lipid, a second helper lipid). A “helper lipid” asprovided herein refers to a lipid capable of increasing delivery of thebioactive agent to a cell relative to the absence of the helper lipid.Thus, the delivery efficiency of a bioactive agent to a cell is higherin the presence of a helper lipid relative to the delivery efficiency inthe absence of said helper lipid. Delivery of a bioactive agent into acell includes, for example, uptake of the bioactive agent into a cell(penetration through the cell membrane), endosomal release of thebioactive agent in a cell, enhancing stability of the bioactive agentand/or the compounds forming the lipid aggregate during the process ofdelivery. Helper lipids useful in this invention include, withoutlimitation: lecithins; phosphotidylethanolamine;phosphatidylethanolamines, such as DOPE(dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine), DPPE(dipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidylethanolamine, POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, such as DOPC (dioleoylphosphidylcholine), DPPC(dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidylglycerol; phosphatidylglycerols, such as DOPG(dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidyl-glycerol),and distearoylphosphatidylglycerol; phosphatidylserine;phosphatidylserines, such as dioleoyl- or dipalmitoylphosphatidylserine;diphosphatidylglycerols; fatty acid esters; glycerol esters;sphingolipids; cardolipin; cerebrosides; and ceramides; and mixturesthereof. Helper lipids also include cholesterol and other 3βOH-sterols.

A “biostability enhancing agent” as provided herein is a compoundcapable of increasing the physical and chemical stability of thecompositions (e.g., lipid aggregates including a bioactive agent)provided herein relative to the absence of the compound. Uponadministration to a subject a biostability enhancing agent may increasethe biodistribution of the compounds provided herein. In embodiments,the biostability enhancing agent is a polyether compound. Inembodiments, the biostability enhancing agent is a PEGylatedphospholipid. In embodiments, the biostability enhancing agent ispolyethylene glycol.

In embodiments, the first cationic lipid is present at a compositionalmolar ratio of about 0.18. In embodiments, the first cationic lipid ispresent at a compositional molar ratio of 0.18. In embodiments, thefirst cationic lipid is present at a compositional molar ratio of about0.23. In embodiments, the first cationic lipid is present at acompositional molar ratio of 0.23. In embodiments, the first cationiclipid is present at a compositional molar ratio of about 0.24. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio of 0.24. In embodiments, the first cationic lipid is presentat a compositional molar ratio of about 0.25. In embodiments, the firstcationic lipid is present at a compositional molar ratio of 0.25. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio of about 0.27. In embodiments, the first cationic lipid ispresent at a compositional molar ratio of 0.27. In embodiments, thefirst cationic lipid is present at a compositional molar ratio of about0.28. In embodiments, the first cationic lipid is present at acompositional molar ratio of 0.28. In embodiments, the first cationiclipid is present at a compositional molar ratio of about 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio of 0.32.

In embodiments, the first cationic lipid has the formula:

whereinX_(a) ⁻ is Cl⁻ or CH₃COO⁻. In embodiments, X_(a) ⁻ is CH₃COO⁻. Inembodiments, the first cationic lipid is dihydroxy dimyristylspermidine.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio of about 0.24. In embodiments, the second cationic lipid ispresent at a compositional molar ratio of 0.24. In embodiments, thesecond cationic lipid is present at a compositional molar ratio of about0.38. In embodiments, the second cationic lipid is present at acompositional molar ratio of 0.38. In embodiments, the second cationiclipid is present at a compositional molar ratio of about 0.39. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio of 0.39. In embodiments, the second cationic lipid ispresent at a compositional molar ratio of about 0.40. In embodiments,the second cationic lipid is present at a compositional molar ratio of0.40. In embodiments, the second cationic lipid is present at acompositional molar ratio of about 0.45. In embodiments, the secondcationic lipid is present at a compositional molar ratio of 0.45. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio of about 0.47. In embodiments, the second cationic lipid ispresent at a compositional molar ratio of 0.47. In embodiments, thesecond cationic lipid is present at a compositional molar ratio of about0.51. In embodiments, the second cationic lipid is present at acompositional molar ratio of 0.51.

In embodiments, the second cationic lipid has the formula:

wherein X_(b) ⁻ is Cl⁻ or CH₃COO⁻. In embodiments, X_(b) ⁻ is CH₃COO⁻.In embodiments, the second cationic lipid is hydroxy dimyristylspermidine.

In embodiments, the first helper lipid is present at a compositionalmolar ratio of about 0.20. In embodiments, the first helper lipid ispresent at a compositional molar ratio of 0.20. In embodiments, thefirst helper lipid is present at a compositional molar ratio of about0.24. In embodiments, the first helper lipid is present at acompositional molar ratio of 0.24. In embodiments, the first helperlipid is present at a compositional molar ratio of about 0.26. Inembodiments, the first helper lipid is present at a compositional molarratio of 0.26. In embodiments, the first helper lipid is present at acompositional molar ratio of about 0.32. In embodiments, the firsthelper lipid is present at a compositional molar ratio of 0.32. Inembodiments, the first helper lipid is dioleoylphosphatidylethanolamine(DOPE).

In embodiments, the second helper lipid is present at a compositionalmolar ratio of about 0.01. In embodiments, the second helper lipid ispresent at a compositional molar ratio of 0.01. In embodiments, thesecond helper lipid is present at a compositional molar ratio of about0.05. In embodiments, the second helper lipid is present at acompositional molar ratio of 0.05. In embodiments, the second helperlipid is present at a compositional molar ratio of about 0.08. Inembodiments, the second helper lipid is present at a compositional molarratio of 0.08. In embodiments, the second helper lipid is present at acompositional molar ratio of about 0.10. In embodiments, the secondhelper lipid is present at a compositional molar ratio of 0.10. Inembodiments, the second helper lipid is present at a compositional molarratio of about 0.14. In embodiments, the second helper lipid is presentat a compositional molar ratio of 0.14. In embodiments, the secondhelper lipid is cholesterol.

In embodiments, the biostability enhancing agent is present at acompositional molar ratio of about 0.01. In embodiments, thebiostability enhancing agent is present at a compositional molar ratioof 0.01. In embodiments, the biostability enhancing agent is present ata compositional molar ratio of about 0.02. In embodiments, thebiostability enhancing agent is present at a compositional molar ratioof 0.02. In embodiments, the biostability enhancing agent is a polyethercompound. In embodiments, the biostability enhancing agent is aPEGylated phospholipid. In embodiments, the biostability enhancing agentis polyethylene glycol.

In embodiments, the biostability enhancing agent has a molecular weightfrom about 750 g/mol to about 5000 g/mol. In embodiments, thebiostability enhancing agent has a molecular weight from about 800/molto about 5000 g/mol. In embodiments, the biostability enhancing agenthas a molecular weight from about 850 g/mol to about 5000 g/mol. Inembodiments, the biostability enhancing agent has a molecular weightfrom about 900 g/mol to about 5000 g/mol. In embodiments, thebiostability enhancing agent has a molecular weight from about 950 g/molto about 5000 g/mol. In embodiments, the biostability enhancing agenthas a molecular weight from about 1000 g/mol to about 5000 g/mol. Inembodiments, the biostability enhancing agent has a molecular weightfrom about 1500 g/mol to about 5000 g/mol. In embodiments, thebiostability enhancing agent has a molecular weight from about 2000g/mol to about 5000 g/mol. In embodiments, the biostability enhancingagent has a molecular weight from about 2500 g/mol to about 5000 g/mol.In embodiments, the biostability enhancing agent has a molecular weightfrom about 3000 g/mol to about 5000 g/mol. In embodiments, thebiostability enhancing agent has a molecular weight from about 3500g/mol to about 5000 g/mol. In embodiments, the biostability enhancingagent has a molecular weight from about 4000 g/mol to about 5000 g/mol.In embodiments, the biostability enhancing agent has a molecular weightfrom about 4500 g/mol to about 5000 g/mol.

In embodiments, the biostability enhancing agent has a molecular weightof about 750 g/mol. In embodiments, the biostability enhancing agent hasa molecular weight of 750 g/mol. In embodiments, the biostabilityenhancing agent has a molecular weight of about 2000 g/mol. Inembodiments, the biostability enhancing agent has a molecular weight of2000 g/mol. In embodiments, the biostability enhancing agent has amolecular weight of about 5000 g/mol. In embodiments, the biostabilityenhancing agent has a molecular weight of 5000 g/mol.

In embodiments, the biostability enhancing agent is C14 polyethyleneglycol 750. In embodiments, the biostability enhancing agent is C14polyethylene glycol 2000. In embodiments, biostability enhancing agentis C14 polyethylene glycol 5000.

In embodiments, the first cationic lipid is present at a compositionalmolar ratio from about 0.18 to about 0.32, from about 0.19 to about0.32, from about 0.20 to about 0.32, from about 0.21 to about 0.32, fromabout 0.22 to about 0.32, from about 0.23 to about 0.32, from about 0.24to about 0.32, from about 0.25 to about 0.32, from about 0.26 to about0.32, from about 0.27 to about 0.32, from about 0.28 to about 0.32, fromabout 0.29 to about 0.32, or from about 0.30 to about 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.32, from 0.19 to 0.32, from 0.20 to 0.32,from 0.21 to 0.32, from 0.22 to 0.32, from 0.23 to 0.32, from 0.24 to0.32, from 0.25 to 0.32, from 0.26 to 0.32, from 0.27 to 0.32, from 0.28to 0.32, from 0.29 to 0.32, or from 0.30 to 0.32.

In embodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.32. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.19 to 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.20 to 0.32. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.21 to 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.22 to 0.32. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.23 to 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.24 to 0.32. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.25 to 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.26 to 0.32. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.27 to 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.28 to 0.32. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.29 to 0.32. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.30 to 0.32.

In embodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.31. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.18 to 0.30. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.29. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.18 to 0.28. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.27. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.18 to 0.26. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.25. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.18 to 0.24. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.23. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.18 to 0.22. Inembodiments, the first cationic lipid is present at a compositionalmolar ratio from 0.18 to 0.21. In embodiments, the first cationic lipidis present at a compositional molar ratio from 0.18 to 0.20.

In embodiments, the first cationic lipid is present at a compositionalmolar ratio of 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26,0.27, 0.28, 0.29, 0.30, 0.31 or 0.32. In embodiments, the first cationiclipid is present at a compositional molar ratio of about 0.18, 0.19,0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31or 0.32. In further embodiments, the second cationic lipid is present ata compositional molar ratio from about 0.24 to about 0.51, the firsthelper lipid is present at a compositional molar ratio from about 0.20to about 0.32, the second helper lipid is present at a compositionalmolar ratio from about 0.01 to about 0.14 and the biostability enhancingagent is present at a compositional molar ratio from about 0.01 to about0.02.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio from about 0.24 to about 0.51, from about 0.25 to about0.51, from about 0.26 to about 0.51, from about 0.27 to about 0.51, fromabout 0.28 to about 0.51, from about 0.29 to about 0.51, from about 0.30to about 0.51, from about 0.31 to about 0.51, from about 0.32 to about0.51, from about 0.33 to about 0.51, from about 0.34 to about 0.51, fromabout 0.35 to about 0.51, from about 0.36 to about 0.51, from about 0.37to about 0.51, from about 0.38 to about 0.51, from about 0.39 to about0.51, from about 0.40 to about 0.51, from about 0.41 to about 0.51, fromabout 0.42 to about 0.51, from about 0.43 to about 0.51, from about 0.44to about 0.51, from about 0.45 to about 0.51, from about 0.46 to about0.51, from about 0.47 to about 0.51, from about 0.48 to about 0.51, orfrom about 0.49 to about 0.51.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.24 to 0.51, from 0.25 to 0.51, from 0.26 to 0.51,from 0.27 to 0.51, from 0.28 to 0.51, from 0.29 to 0.51, from 0.30 to0.51, from 0.31 to 0.51, from 0.32 to 0.51, from 0.33 to 0.51, from 0.34to 0.51, from 0.35 to 0.51, from 0.36 to 0.51, from 0.37 to 0.51, from0.38 to 0.51, from 0.39 to 0.51, from 0.40 to 0.51, from 0.41 to 0.51,from 0.42 to 0.51, from 0.43 to 0.51, from 0.44 to 0.51, from 0.45 to0.51, from 0.46 to 0.51, from 0.47 to 0.51, from 0.48 to 0.51, or from0.49 to 0.51.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio from about 0.24 to about 0.51, from about 0.24 to about0.50, from about 0.24 to about 0.49, from about 0.24 to about 0.48, fromabout 0.24 to about 0.47, from about 0.24 to about 0.46, from about 0.24to about 0.45, from about 0.24 to about 0.44, from about 0.24 to about0.43, from about 0.24 to about 0.42, from about 0.24 to about 0.41, fromabout 0.24 to about 0.40, from about 0.24 to about 0.39, from about 0.24to about 0.38, from about 0.24 to about 0.37, from about 0.24 to about0.36, from about 0.24 to about 0.35, from about 0.24 to about 0.34, fromabout 0.24 to about 0.33, from about 0.24 to about 0.32, from about 0.24to about 0.31, from about 0.24 to about 0.30, from about 0.24 to about0.29, from about 0.24 to about 0.28, from about 0.24 to about 0.27, orfrom about 0.24 to about 0.26.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.24 to 0.51, from 0.24 to 0.50, from 0.24 to 0.49,from 0.24 to 0.48, from 0.24 to 0.47, from 0.24 to 0.46, from 0.24 to0.45, from 0.24 to 0.44, from 0.24 to 0.43, from 0.24 to 0.42, from 0.24to 0.41, from 0.24 to 0.40, from 0.24 to 0.39, from 0.24 to 0.38, from0.24 to 0.37, from 0.24 to 0.36, from 0.24 to 0.35, from 0.24 to 0.34,from 0.24 to 0.33, from 0.24 to 0.32, from 0.24 to 0.31, from 0.24 to0.30, from 0.24 to 0.29, from 0.24 to 0.28, from 0.24 to 0.27, or from0.24 to 0.26.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.24 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.25 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.26 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.27 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.28 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.29 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.30 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.31 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.32 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.33 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.34 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.35 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.36 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.37 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.38 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.39 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.40 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.41 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.42 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.43 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.44 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.45 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.46 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio from 0.47 to 0.51. Inembodiments, the second cationic lipid is present at a compositionalmolar ratio from 0.48 to 0.51. In embodiments, the second cationic lipidis present at a compositional molar ratio or from 0.49 to 0.51.

In embodiments, the second cationic lipid is present at a compositionalmolar ratio of 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32,0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44,0.45, 0.46, 0.47, 0.48, 0.49, 0.50 or 0.51. In embodiments, the secondcationic lipid is present at a compositional molar ratio of about 0.24,0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36,0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48,0.49, 0.50 or 0.51. In further embodiments, the first cationic lipid ispresent at a compositional molar ratio from about 0.18 to about 0.32,the first helper lipid is present at a compositional molar ratio fromabout 0.20 to about 0.32, the second helper lipid is present at acompositional molar ratio from about 0.01 to about 0.14 and thebiostability enhancing agent is present at a compositional molar ratiofrom about 0.01 to about 0.02.

In embodiments, the first helper lipid is present at a compositionalmolar ratio from 0.20 to 0.32, from 0.21 to 0.32, from 0.22 to 0.32,from 0.23 to 0.32, from 0.24 to 0.32, from 0.25 to 0.32, from 0.26 to0.32, from 0.27 to 0.32, from 0.28 to 0.32, from 0.29 to 0.32, or from0.30 to 0.32. In embodiments, the first helper lipid is present at acompositional molar ratio from about 0.20 to about 0.32, from about 0.21to about 0.32, from about 0.22 to about 0.32, from about 0.23 to about0.32, from about 0.24 to about 0.32, from about 0.25 to about 0.32, fromabout 0.26 to about 0.32, from about 0.27 to about 0.32, from about 0.28to about 0.32, from about 0.29 to about 0.32, or from about 0.30 toabout 0.32.

In embodiments, the first helper lipid is present at a compositionalmolar ratio from 0.20 to 0.31, from 0.20 to 0.30, from 0.20 to 0.29,from 0.20 to 0.28, from 0.20 to 0.27, from 0.20 to 0.26, from 0.20 to0.25, from 0.20 to 0.24, from 0.20 to 0.23, or from 0.20 to 0.22. Inembodiments, the first helper lipid is present at a compositional molarratio from about 0.20 to about 0.31, from about 0.20 to about 0.30, fromabout 0.20 to about 0.29, from about 0.20 to about 0.28, from about 0.20to about 0.27, from about 0.20 to about 0.26, from about 0.20 to about0.25, from about 0.20 to about 0.24, from about 0.20 to about 0.23, orfrom about 0.20 to about 0.22.

In embodiments, the first helper lipid is present at a compositionalmolar ratio from 0.20 to 0.31. In embodiments, the first helper lipid ispresent at a compositional molar ratio from 0.20 to 0.30. Inembodiments, the first helper lipid is present at a compositional molarratio from 0.20 to 0.29. In embodiments, the first helper lipid ispresent at a compositional molar ratio from 0.20 to 0.28. Inembodiments, the first helper lipid is present at a compositional molarratio from 0.20 to 0.27. In embodiments, the first helper lipid ispresent at a compositional molar ratio from 0.20 to 0.26. Inembodiments, the first helper lipid is present at a compositional molarratio from 0.20 to 0.25. In embodiments, the first helper lipid ispresent at a compositional molar ratio from 0.20 to 0.24. Inembodiments, the first helper lipid is present at a compositional molarratio from 0.20 to 0.23. In embodiments, the first helper lipid ispresent at a compositional molar ratio or from 0.20 to 0.22.

In embodiments, the first helper lipid is present at a compositionalmolar ratio from 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28,0.29, 0.30, 0.31, or 0.32. In embodiments, the first helper lipid ispresent at a compositional molar ratio from about 0.20, 0.21, 0.22,0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, or 0.32. Infurther embodiments, the first cationic lipid is present at acompositional molar ratio from about 0.18 to about 0.32, the secondcationic lipid is present at a compositional molar ratio from about 0.24to about 0.51, the second helper lipid is present at a compositionalmolar ratio from about 0.01 to about 0.14 and the biostability enhancingagent is present at a compositional molar ratio from about 0.01 to about0.02.

In embodiments, the second helper lipid is present at a compositionalmolar ratio from 0.01 to 0.14, from 0.01 to 0.13, from 0.01 to 0.12,from 0.01 to 0.11, from 0.01 to 0.10, from 0.01 to 0.9, from 0.01 to0.8, from 0.01 to 0.7, from 0.01 to 0.6, from 0.01 to 0.5, from 0.01 to0.4, from 0.01 to 0.3, from 0.01 to 0.2, from 0.01 to 0.1, from 0.01 to0.09, from 0.01 to 0.08, from 0.01 to 0.07, from 0.01 to 0.06, from 0.01to 0.05, from 0.01 to 0.04, or from 0.01 to 0.03. In embodiments, thesecond helper lipid is present at a compositional molar ratio from about0.01 to about 0.14, from about 0.01 to about 0.13, from about 0.01 toabout 0.12, from about 0.01 to about 0.11, from about 0.01 to about0.10, from about 0.01 to about 0.9, from about 0.01 to about 0.8, fromabout 0.01 to about 0.7, from about 0.01 to about 0.6, from about 0.01to about 0.5, from about 0.01 to about 0.4, from about 0.01 to about0.3, from about 0.01 to about 0.2, from about 0.01 to about 0.1, fromabout 0.01 to about 0.09, from about 0.01 to about 0.08, from about 0.01to about 0.07, from about 0.01 to about 0.06, from about 0.01 to about0.05, from about 0.01 to about 0.04, or from about 0.01 to about 0.03.

In embodiments, the second helper lipid is present at a compositionalmolar ratio from 0.02 to 0.14. In embodiments, the second helper lipidis present at a compositional molar ratio from 0.03 to 0.14. Inembodiments, the second helper lipid is present at a compositional molarratio from 0.04 to 0.14. In embodiments, the second helper lipid ispresent at a compositional molar ratio from 0.05 to 0.14. Inembodiments, the second helper lipid is present at a compositional molarratio from 0.06 to 0.14. In embodiments, the second helper lipid ispresent at a compositional molar ratio from 0.07 to 0.14. Inembodiments, the second helper lipid is present at a compositional molarratio from 0.08 to 0.14. In embodiments, the second helper lipid ispresent at a compositional molar ratio from 0.09 to 0.14. Inembodiments, the second helper lipid is present at a compositional molarratio from 0.1 to 0.14. In embodiments, the second helper lipid ispresent at a compositional molar ratio from 0.2 to 0.14. In embodiments,the second helper lipid is present at a compositional molar ratio from0.3 to 0.14. In embodiments, the second helper lipid is present at acompositional molar ratio from 0.4 to 0.14. In embodiments, the secondhelper lipid is present at a compositional molar ratio from 0.5 to 0.14.In embodiments, the second helper lipid is present at a compositionalmolar ratio from 0.6 to 0.14. In embodiments, the second helper lipid ispresent at a compositional molar ratio from 0.7 to 0.14. In embodiments,the second helper lipid is present at a compositional molar ratio from0.8 to 0.14. In embodiments, the second helper lipid is present at acompositional molar ratio from 0.9 to 0.14. In embodiments, the secondhelper lipid is present at a compositional molar ratio from 0.1 to 0.14.In embodiments, the second helper lipid is present at a compositionalmolar ratio from 0.11 to 0.14. In embodiments, the second helper lipidis present at a compositional molar ratio from 0.12 to 0.14.

In embodiments, the second helper lipid is present at a compositionalmolar ratio from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.10, 0.11, 0.12, 0.13, or 0.14. In embodiments, the second helper lipidis present at a compositional molar ratio from about 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, or 0.14. Infurther embodiments, the first cationic lipid is present at acompositional molar ratio from about 0.18 to about 0.32, the secondcationic lipid is present at a compositional molar ratio from about 0.24to about 0.51, the first helper lipid is present at a compositionalmolar ratio from about 0.20 to about 0.32 and the biostability enhancingagent is present at a compositional molar ratio from about 0.01 to about0.02.

In embodiments, the biostability enhancing agent is present at acompositional molar ratio from 0.01 to 0.02. In embodiments, thebiostability enhancing agent is present at a compositional molar ratiofrom 0.011 to 0.02. In embodiments, the biostability enhancing agent ispresent at a compositional molar ratio from 0.012 to 0.02. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from 0.013 to 0.02. In embodiments, thebiostability enhancing agent is present at a compositional molar ratiofrom 0.014 to 0.02. In embodiments, the biostability enhancing agent ispresent at a compositional molar ratio from 0.015 to 0.02. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from 0.016 to 0.02. In embodiments, thebiostability enhancing agent is present at a compositional molar ratiofrom 0.017 to 0.02. In embodiments, the biostability enhancing agent ispresent at a compositional molar ratio from 0.018 to 0.02.

In embodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.02. In embodiments,the biostability enhancing agent is present at a compositional molarratio from about 0.011 to about 0.02. In embodiments, the biostabilityenhancing agent is present at a compositional molar ratio from about0.012 to about 0.02. In embodiments, the biostability enhancing agent ispresent at a compositional molar ratio from about 0.013 to about 0.02.In embodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.014 to about 0.02. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.015 to about 0.02. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.016 to about 0.02. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.017 to about 0.02. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.018 to about 0.02.

In embodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.019. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.018. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.017. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.016. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.015. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.014. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.013. Inembodiments, the biostability enhancing agent is present at acompositional molar ratio from about 0.01 to about 0.012.

In embodiments, the biostability enhancing agent is present at acompositional molar ratio from 0.01, 0.011, 0.012, 0.013, 0.014, 0.015,0.016, 0.017, 0.018, 0.019 or 0.020. In embodiments, the biostabilityenhancing agent is present at a compositional molar ratio from about0.01, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019 or0.020. In further embodiments, the first cationic lipid is present at acompositional molar ratio from about 0.18 to about 0.32, the secondcationic lipid is present at a compositional molar ratio from about 0.24to about 0.51, the first helper lipid is present at a compositionalmolar ratio from about 0.20 to about 0.32 and the second helper lipid ispresent at a compositional molar ratio from about 0.01 to about 0.14.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.24, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.05, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.01, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.32, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.39, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.26, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.01, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.18, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.23, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.45, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.20, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.18, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.51, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.20, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.01, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.27, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.01, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.25, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.26, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.01, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.28, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.24, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.14, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 750.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.18, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.47, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.32, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.01, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein saidbiostability enhancing agent is C14 polyethylene glycol 5000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.24, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.40, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.24, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.10, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 2000.

In an aspect is provided a composition including: (i) a first cationiclipid at a compositional molar ratio of about 0.32, wherein the firstcationic lipid is dihydroxy dimyristyl spermidine; (ii) a secondcationic lipid at a compositional molar ratio of about 0.38, wherein thesecond cationic lipid is hydroxy dimyristyl spermidine; (iii) a firsthelper lipid at a compositional molar ratio of about 0.20, wherein thefirst helper lipid is dioleoylphosphatidylethanolamine; (iv) a secondhelper lipid at a compositional molar ratio of about 0.08, wherein thesecond helper lipid is cholesterol; and (v) a biostability enhancingagent at a compositional molar ratio of about 0.02, wherein thebiostability enhancing agent is C14 polyethylene glycol 5000.

In embodiments, the composition as provided herein including embodimentsthereof, further includes a bioactive agent. A “bioactive agent” asprovided herein refers to a compound that upon administration to a cell,tissue or organism has a detectable effect on the biological function ofsaid cell, tissue or organism. In embodiments, the detectable effect isa biological effect. In embodiments, the detectable effect is atherapeutic effect. In embodiments, the detectable effect is adiagnostic effect. The bioactive agent is capable of forming a lipidaggregate with the compositions provided herein including embodimentsthereof. In embodiments, the bioactive agent is a test compound. A “testcompound” as provided herein is a compound whose effect on a biologicalfunction is determined relative to a control compound. A “controlcompound” as provided herein refers to a compound having a known effecton a biological function. In embodiments, the bioactive agent is acontrol compound. In embodiments, the bioactive agent is a therapeuticagent or a diagnostic agent. In embodiments, the bioactive agent is atherapeutic agent or a diagnostic agent. In embodiments, the bioactiveagent is a therapeutic agent. In embodiments, the bioactive agent is adiagnostic agent. In embodiments, the bioactive agent includes a nucleicacid, a ribonucleoprotein or a small molecule. In embodiments, thebioactive agent includes a nucleic acid. In embodiments, the bioactiveagent includes a ribonucleoprotein. In embodiments, the bioactive agentincludes a small molecule. In embodiments, the nucleic acid is an mRNA,a siRNA, a miRNA or a guide RNA. In embodiments, the nucleic acid is anmRNA. In embodiments, the nucleic acid is a siRNA. In embodiments, thenucleic acid is a miRNA. In embodiments, the nucleic acid is a guideRNA. In embodiments, the bioactive agent includes a nucleic acid and aribonucleoprotein. In embodiments, the ribonucleoprotein is CRISPRassociated protein 9 (Cas9). An “mRNA” as provided herein refers to aribonucleic acid molecule, including one or more than one expressiblenucleic acid sequences encoding one or more proteins or polypeptides, orother DNA molecules.

Pharmaceutical Composition

In an aspect is provided a pharmaceutical composition including acomposition as provided herein including embodiments thereof and apharmaceutically acceptable excipient.

A therapeutically effective amount as provided herein refers to anamount effective to achieve its intended purpose. The actual amounteffective for a particular application will depend, inter alia, on thecondition being treated. When administered in methods to treat adisease, the pharmaceutical compositions described herein will containan amount of active bioactive agent effective to achieve the desiredresult, e.g., modulating the activity of a target molecule and/orreducing, eliminating, or slowing the progression of disease symptoms(e.g., pulmonary disease). Determination of a therapeutically effectiveamount of a bioactive agent forming a lipid aggregate with thecompositions provided herein is well within the capabilities of thoseskilled in the art, especially in light of the detailed disclosureherein.

Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, or acetate at a pH typically of 5.0to 8.0, most often 6.0 to 7.0; salts such as sodium chloride, potassiumchloride, etc. to make isotonic; antioxidants, preservatives, lowmolecular weight polypeptides, proteins, hydrophilic polymers such aspolysorbate 80, amino acids such as glycine, carbohydrates, chelatingagents, sugars, and other standard ingredients known to those skilled inthe art (Remington's Pharmaceutical Science 16^(th) edition, Osol, A.Ed. 1980).

A pharmaceutical composition including a composition provided hereinincluding embodiments thereof (e.g., a lipid aggregate complexed with abioactive agent) can be administered by a variety of methods known inthe art. The route and/or mode of administration vary depending upon thedesired results. In embodiments, administration is intravenous,intramuscular, intraperitoneal, or subcutaneous, or administeredproximal to the site of the target. Pharmaceutically acceptableexcipients can be suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion).

Pharmaceutical compositions of the composition provided herein includingembodiments thereof (e.g., a lipid aggregate complexed with a bioactiveagent) can be prepared in accordance with methods well known androutinely practiced in the art. See, e.g., Remington: The Science andPractice of Pharmacy, Mack Publishing Co., 20^(th) ed., 2000; andSustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositionsare preferably manufactured under GMP conditions. Typically, atherapeutically effective dose or efficacious dose of the compositionprovided herein including embodiments thereof (e.g., a lipid aggregatecomplexed with a bioactive agent) is employed in the pharmaceuticalcompositions of the invention. The composition provided herein includingembodiments thereof (e.g., a lipid aggregate complexed with a bioactiveagent) can be formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art. Dosageregimens are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It may be advantageous to formulate thecomposition provided herein including embodiments thereof (e.g., a lipidaggregate complexed with a bioactive agent) in combination with othertherapies or agents. It can be advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of a compositionprovided herein including embodiments thereof (e.g., a lipid aggregatecomplexed with a bioactive agent) calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalexcipient.

Actual dosage levels of the bioactive agent in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular antibody being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors.

A physician or veterinarian can start doses of the composition providedherein including embodiments thereof (e.g., a lipid aggregate complexedwith a bioactive agent) employed in the pharmaceutical composition atlevels lower than that required to achieve the desired therapeuticeffect and gradually increase the dosage until the desired effect isachieved. In general, effective doses of the compositions of the presentinvention vary depending upon many different factors, including thespecific disease or condition to be treated, means of administration,target site, physiological state of the patient, whether the patient ishuman or an animal, other medications administered, and whethertreatment is prophylactic or therapeutic. Treatment dosages need to betitrated to optimize safety and efficacy.

Composition provided herein including embodiments thereof (e.g., a lipidaggregate complexed with a bioactive agent) can be administered onmultiple occasions. Intervals between single dosages can be weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of the composition in the patient. Dosage andfrequency vary depending on the half-life of the composition in thepatient. The dosage and frequency of administration can vary dependingon whether the treatment is prophylactic or therapeutic. In prophylacticapplications, a relatively low dosage is administered at relativelyinfrequent intervals over a long period of time. Some patients continueto receive treatment for the rest of their lives. In therapeuticapplications, a relatively high dosage at relatively short intervals issometimes required until progression of the disease is reduced orterminated, and preferably until the patient shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

Cellular Compositions

In an aspect is provided a cell including a composition as providedherein including embodiments thereof. In embodiments, the cell is amammalian cell. In embodiments, the cell is a rodent cell. Inembodiments, the cell is a mouse cell. In embodiments, the cell is a ratcell. In embodiments, the cell is a porcine cell. In embodiments, thecell is a canine cell. In embodiments, the cell is a primate cell. Inembodiments, the cell is a human cell. In embodiments, the cell is anepithelial cell. In embodiments, the cell is an epithelial lung cell. Inembodiments, the cell is an endothelial cell. In embodiments, the cellis an endothelial lung cell. In embodiments, the cell forms part of anorganism. In embodiments, the organism is human. In embodiments, theorganism is rat. In embodiments, the organism is mouse.

Methods of Delivery

In an aspect is provided, method of delivering a bioactive agent to acell, the method including: (i) admixing an bioactive agent with acomposition as provided herein including embodiments thereof, therebyforming a bioactive agent-lipid complex; (ii) contacting a cell with thebioactive agent-lipid complex, thereby delivering the bioactiveagent-lipid complex to a cell. The bioactive agent may be any bioactiveagent as described herein (e.g. a nucleic acid). The bioactiveagent-lipid complex as provided herein is a lipid aggregate as describedherein.

In embodiments, the method as described herein including embodimentsthereof, further includes allowing the bioactive agent-lipid complex toenter the cell. In embodiments, the bioactive agent is a therapeuticagent or a diagnostic agent. In embodiments, the bioactive agentincludes a nucleic acid, a ribonucleoprotein or a small molecule. Inembodiments, the nucleic acid is an mRNA, a siRNA, miRNA or guide RNA.In embodiments, the bioactive agent includes a guide RNA and aribonucleoprotein. In embodiments, the ribonucleoprotein is CRISPRassociated protein 9 (Cas9). In embodiments, the ribonucleoprotein isbound to the guide RNA.

In embodiments, the cell is a mammalian cell. In embodiments, the cellis a rodent cell. In embodiments, the cell is a mouse cell. Inembodiments, the cell is a rat cell. In embodiments, the cell is aporcine cell. In embodiments, the cell is a canine cell. In embodiments,the cell is a primate cell. In embodiments, the cell is an epithelialcell. In embodiments, the cell is an epithelial lung cell. Inembodiments, the cell is an endothelial cell. In embodiments, the cellis an endothelial lung cell.

In another aspect is provided a method of delivering a bioactive agentto lung tissue in a subject, the method including: (i) admixing anbioactive agent with a composition as described herein includingembodiments thereof, thereby forming a bioactive agent-lipid complex;(ii) systemically administering an effective amount of the bioactiveagent-lipid complex to a subject, thereby delivering the bioactiveagent-lipid complex to a lung tissue in a subject.

In another aspect is provided a method of expressing a protein in lungtissue in a subject, the method including: (i) admixing a mRNA with acomposition as described herein including embodiments thereof, therebyforming a mRNA-lipid complex; (ii) administering an effective amount ofthe mRNA-lipid complex to a subject; and (iii) allowing the mRNA of themRNA-lipid complex to express in lung tissue of the subject, therebyexpressing a protein in lung tissue in a subject.

Methods of Treatment

In an aspect is provided a method of treating a pulmonary disease in asubject in need thereof, the method including administering to a subjecta therapeutically effective amount of a bioactive agent and acomposition as described herein including embodiments thereof, therebytreating a pulmonary disease in the subject.

In embodiments, the composition and the bioactive agent are admixedprior to the administering. In embodiments, bioactive agent includes anucleic acid, a ribonucleoprotein or a small molecule. In embodiments,the nucleic acid is an mRNA, a siRNA, a miRNA or a guide RNA.

In embodiments, the pulmonary disease is asthma, chronic obstructivepulmonary disease (COPD), lung cancer or cystic fibrosis. Inembodiments, the pulmonary disease is asthma. In embodiments, thepulmonary disease is chronic obstructive pulmonary disease (COPD). Inembodiments, the pulmonary disease is lung cancer. In embodiments, thepulmonary disease is or cystic fibrosis.

The terms “pulmonary disease,” “pulmonary disorder,” “lung disease,”etc. are used interchangeably herein. The term is used to broadly referto lung disorders characterized by difficulty breathing, coughing,airway discomfort and inflammation, increased mucus, and/or pulmonaryfibrosis.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. For the present invention, the dose will generally referto the amount of pulmonary disease treatment. The dose will varydepending on a number of factors, including the range of normal dosesfor a given therapy, frequency of administration; size and tolerance ofthe individual; severity of the condition; risk of side effects; and theroute of administration. One of skill will recognize that the dose canbe modified depending on the above factors or based on therapeuticprogress. The term “dosage form” refers to the particular format of thepharmaceutical, and depends on the route of administration. For example,a dosage form can be in a liquid form for nebulization, e.g., forinhalants, or a saline solution, e.g., for injection.

As used herein, the terms “treat” and “prevent” are not intended to beabsolute terms. Treatment can refer to any delay in onset, reduction inthe frequency or severity of symptoms, amelioration of symptoms,improvement in patient comfort and/or respiratory function, etc. Theeffect of treatment can be compared to an individual or pool ofindividuals not receiving a given treatment, or to the same patientprior to, or after cessation of, treatment.

“Treating” or “treatment” as used herein (and as well-understood in theart) also broadly includes any approach for obtaining beneficial ordesired results in a subject's condition, including clinical results.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions,diminishment of the extent of a disease, stabilizing (i.e., notworsening) the state of disease, prevention of a disease's transmissionor spread, delay or slowing of disease progression, amelioration orpalliation of the disease state, diminishment of the reoccurrence ofdisease, and remission, whether partial or total and whether detectableor undetectable. In other words, “treatment” as used herein includes anycure, amelioration, or prevention of a disease. Treatment may preventthe disease from occurring; inhibit the disease's spread; relieve thedisease's symptoms (e.g., ocular pain, seeing halos around lights, redeye, very high intraocular pressure), fully or partially remove thedisease's underlying cause, shorten a disease's duration, or do acombination of these things.

“Treating” and “treatment” as used herein include prophylactictreatment. Treatment methods include administering to a subject atherapeutically effective amount of an active agent. The administeringstep may consist of a single administration or may include a series ofadministrations. The length of the treatment period depends on a varietyof factors, such as the severity of the condition, the age of thepatient, the concentration of active agent, the activity of thecompositions used in the treatment, or a combination thereof. It willalso be appreciated that the effective dosage of an agent used for thetreatment or prophylaxis may increase or decrease over the course of aparticular treatment or prophylaxis regime. Changes in dosage may resultand become apparent by standard diagnostic assays known in the art. Insome instances, chronic administration may be required. For example, thecompositions are administered to the subject in an amount and for aduration sufficient to treat the patient.

The term “prevent” refers to a decrease in the occurrence of pulmonarydisease symptoms in a patient. As indicated above, the prevention may becomplete (no detectable symptoms) or partial, such that fewer symptomsare observed than would likely occur absent treatment.

The term “therapeutically effective amount,” as used herein, refers tothat amount of the therapeutic agent sufficient to ameliorate thedisorder, as described above. For example, for the given parameter, atherapeutically effective amount will show an increase or decrease of atleast 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least100%. Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol.

The term “diagnosis” refers to a relative probability that a pulmonarydisease is present in the subject. Similarly, the term “prognosis”refers to a relative probability that a certain future outcome may occurin the subject. For example, in the context of the present invention,prognosis can refer to the likelihood that an individual will develop apulmonary disease, or the likely severity of the disease (e.g., severityof symptoms, rate of functional decline, survival, etc.). The terms arenot intended to be absolute, as will be appreciated by any one of skillin the field of medical diagnostics.

EXAMPLES Example 1: Novel Lipid Nanoparticles for In Vivo Lung TargetedmRNA and siRNA Delivery

The rapidly expanding utilization of nucleic acids as a therapeutic toolhas presented the field with the task of optimizing and innovatingdelivery methods. Lipid nanoparticles are a common delivery vehicle dueto their ability to facilitate cellular uptake while protecting thepayload from extracellular enzyme degradation. Organ and tissue specificdelivery of sensitive payloads such as mRNA is of great importance intailoring therapeutic functionality. This is commonly achieved usingbiomolecular targeting via ligand or receptor expression on the surfaceof the nanoparticle. However, manipulation of the inherent properties ofnanoparticles affords the opportunity to tailor the location of deliveryto a specific organ of interest.

Applicants have developed novel lipid nanoparticles that arespecifically optimized for use in vivo, and engineered to inherentlytarget and deliver nucleic acid payloads (mRNA and siRNA) to murinelungs and spleen without the use of biomolecular targeting. Developmentof the lipid nanoparticle was performed using multivariable Design ofExperiment modeling, which allowed Applicants to understand and optimizethe lipid formulation components and composition. Characterization ofthe lipid nanoparticles showed uniform size and high encapsulationefficiency. Following multiple design iterations, in vivo functionaltesting to assess biodistribution identified a novel formulation capableof exclusively targeting lung tissue and achieving highly efficient mRNAtransfection. Transfection efficiency was measured following in vivosystemic delivery of a chemically modified luciferase encoding mRNA.Quantification was performed using the IVIS imaging system to measure invivo and ex vivo bioluminescence measurements.

Example 2: Development of Novel Lipid Nanoparticles for In Vivo LungTargeted mRNA Delivery

Abstract.

The rapidly expanding utilization of mRNA as a therapeutic tool haspresented the field with the task of optimizing and innovating deliverymethods. As the applications for RNA based therapeutics continues torise, a parallel emerging need to improve upon and develop noveltechnology has come to the forefront. Lipid nanoparticles area commondelivery vehicle for mRNA due to their ability to facilitate cellularuptake while protecting the mRNA from extracellular enzyme degradation.Organ and tissue specific delivery of mRNA is of great importance intailoring therapeutic functionality. This is commonly achieved usingbiomolecular targeting via ligand or receptor expression on the surfaceof the nanoparticle.

Applicants have developed novel lipid nanoparticles that arespecifically optimized for use in vivo, and engineered to inherentlytarget and deliver mRNA to murine lungs and spleen without the use ofbiomolecular targeting. Development of the lipid nanoparticle wasperformed using multivariable Design of Experiment modeling, whichallowed Applicants to understand and optimize the lipid formulationcomponents and composition. Characterization of the lipid nanoparticlesshowed uniform size and high encapsulation efficiency. Followingmultiple design iterations, in vivo functional testing to assessbiodistribution identified a novel formulation capable of exclusivelytargeting the lung tissue and achieving highly efficient mRNAtransfection. Transfection efficiency was measured following in vivosystemic delivery of a chemically modified luciferase encoding mRNA.Quantification was performed using the IVIS imaging system to measure invivo and ex vivo bioluminescence measurements. Lung specific expressionlevels could be modulated by varying the dose of mRNA, and significantprotein expression was sustained over the course of 48 hours following asingle administration. This novel lipid nanoparticle is well toleratedin vivo, with no qualitative gross toxicity, and quantitatively analyzedby a comprehensive cytokine profiling performed on murine serum samples.Further optimization of biodistribution to achieve exclusive targetingto the lung is currently underway, and preliminary data indicates thatvariance in charge ratio enhances delivery and expression exclusively tothe lung, and depletes expression in other organs.

Introduction.

Due to the unstable nature of mRNA, protection against degradationduring the delivery phase is a serious challenge to overcome in thefield of mRNA therapeutics. Applicants have developed efficient lipidnanoparticle (LNP) systems that encapsulate and protect the mRNA fromdegradation and clearance in the blood; thus translating to improvedefficacy and reduced toxicity in vivo. Optimization of Applicant's LNPsystems has maximized delivery efficiency with organ specific uptakepatterns such as the lung and spleen via intravenous delivery.

Materials and Methods.

Based on mixture Design of Experiment optimization, new LNP formulationswere made, complexed with mRNA using the previously developed protocolshown in FIGS. 1A-1C, and the subsequent LNPs were screened for deliveryin vivo using a luciferase readout. Top performing formulations werethen used to model and predict second generation formulations foroptimized tissue specific expression. These new formulations were thentested in vivo to identify the best LNP formulation.

Firefly Luciferase mRNA was complexed with each LNP formulation andinjected intravenously (FIG. 2). Following 4 hours incubation, Luciferinsubstrate was injected IP, and luciferase expression was measured invivo and ex vivo via the IVIS Lumina LT. Bioluminescence (p/sec/cm̂2/sr)was quantified with Living Image® Software (FIG. 2).

Results.

First pass screening of LNPs revealed organ specific delivery patternsbased on formulation composition or N/P ratio variance. Whole animal invivo imaging indicated organ specific patterns (FIGS. 3A-3B), and exvivo analysis allowed for extensive evaluation of biodistributionpatterns (FIG. 3C-3E). Using the performance data from the firstgeneration of LNPs, expression was optimized using DoE design tomaximize tissue specific performance, focusing mainly on enhancement ofexpression in the spleen and lung.

Over 100 new LNP formulations, spanning 3 generations of optimizationwere screened for performance. For screening, mRNA encoding for fireflyluciferase was complexed with each newly designed formulation andinjected intravenously. Ex vivo quantification of bioluminescence signalin isolated lung tissue (FIG. 4A) or isolated spleen tissue (FIG. 4B)from BALB/c mice, 4-hrs post IV injection is summarized in the graphs.The bioluminescence (p/sec/cm̂2/sr) was quantified using Living Image®Software.

Dose titration of mRNA was performed, and luciferase expression was usedas a readout to measure protein expression. Mice were injected at Time 0h, and imaged over the course of 120 hours (5 days). In vivo images, andcorresponding quantification at 4 hours is shown in FIGS. 5A-5B.Sustained luciferase activity measurement were repeated over theduration of the experiment on the same mice; measurements were takenusing the whole body in vivo imaging technique which is reflected by thelower starting signal values compared to ex vivo measurements. Thebioluminescence (p/sec/cm̂2/sr) was quantified using Living Image®Software and is summarized in the graph in FIG. 5C.

Following optimization of mRNA dose, a time course of luciferaseexpression levels was performed and performance was measured followingremoval of the lung tissue and performing ex vivo measurements over thecourse of 96 hours (FIGS. 6A-6C). Mice were injected intravenously withthe LNPs, whole body imaging was performed at each specified time point,and then ex vivo measurements were obtained on isolated lung tissue.

Quantification of circulating cytokine levels in mouse serum at 2 hrs, 4hrs, 24 hrs and 48 hrs post-injection under two mRNA dose conditions:low (1 mg/kg) and high (3 mg/kg) (FIGS. 7A-7J).

Preliminary results indicate that varying N/P ratio of the LNPs can leadto exclusion of protein expression from certain organs, resulting in atissue specific biodistribution of mRNA (FIG. 8A-8B).

CONCLUSION

LNP formulations were extensively optimized for targeted lung deliveryusing Design of Experiment based testing. Through in vivo systemicdelivery, high fidelity luciferase expression was achieved in lungtissue. Transient expression was sustained for 48 hours posttransfection. Modulation of expression was achieved by varying dose ofmRNA.

Expression in spleen tissue was also achieved, and specificity ofdelivery is currently being optimized through variance of N/P ratio.Ongoing and future studies involve: Determination of transfectionefficiency of specific cell type populations within the targeted organof interest, Increase single organ specificity following systemicdelivery, Stability of LNP formulation before and after mRNAcomplexation.

Example 3: Preparation and Complexation

Formulation Preparation.

Prepare stock solutions of the individual lipid components in 100%Ethanol (EtOH) at appropriate concentrations based on individual lipidsolubility. Heat to 50° C. to dissolve lipids completely in the EtOH. Ina glass vial, combine all 5 lipid components using a pipette in volumescalculated according to Molar ratios listed in Table 1. Dilute thecombined lipids using 200 mM Sodium Acetate at a dilution ratio of 1:4by volume. Seal lid with paraffin. Store at 4° C.

Complexation Preparation.

Concentration of lipid is delivered at a 3 mg/kg concentration.Concentration of mRNA is a 10:1 dilution, so 0.3 mg/mL total mRNA areadministered. Label 2 screw-cap plastic sample tubes as ‘lipid’ and‘mRNA’. Transfer 100 μL of prepared lipid formulation to tube labeled‘lipid’. Calculate amount of mRNA needed for 200 μL total volume ofcomplex. (0.6 mg/mL) Since lipid formulation is at a 25% EtOH solutionfollowing the 1:4 dilution with Sodium Acetate, the mRNA needs to bediluted in a 25% EtOH solution. Add 25 μL of 100% EtOH to tube labeled‘mRNA’. Add appropriate volume of mRNA to ‘mRNA’ tube according tocalculations. Bring up the volume of sample to 100 uL using sterilefiltered water. Add contents of mRNA tube to lipid tube mixing well.Vortex briefly. Heat complex at 50° C. for 30 minutes. Transfer complexto dialysis columns with an 8-10 kD molecular weight cut off, and allowto dialyze for 2 hours in 100% PBS. Transfer sample to air-tight screwcapped sample tube and either use immediately or store at −20° C. untiluse.

Complex Administration.

If using frozen complex, thaw to room temperature before use. Accordingto the institutional guidelines for the care and use of laboratoryanimals, complex is administered to mouse via tail vein injection.

Preparation of formulations was performed as follows. Individual powderlipid components were resuspended in 100% Ethanol (EtOH). Stocksolutions were prepared in the following concentrations: DHDMS at 50mg/mL, HDMS at 15 mg/mL, Cholesterol at 25 mg/mL, DOPE at 50 mg/mL andC14 PEG 5000 (Avanti Polar Lipids, #880210) at 100 mg/mL. Lipidsolutions were heated to 50° C. using a heat block to achieve completesolubility during formulation preparation.

The 5 formulation components were combined in a glass screw top vialusing a pipette in the volumes listed in Table 1, calculated based onthe desired molar ratio and the stock concentration of the lipid. Onceall five of the components were added at the appropriate volumes to theglass vial, the volume was brought up to 100 μL using 100% EtOH. Thisvolume was then diluted at a ratio of 1:4 using a 200 mM Sodium Acetatesolution. The resulting solution was used as the transfection reagent,and stored at 4° C.

Preparation of the lipid/mRNA complexes was performed as follows. Thefinal target concentration of 0.3 mg/mL of mRNA was chosen as thedesired concentration for delivery, and the basis of the calculationsfor sample preparation. 2 screw-cap plastic sample tubes were labeled as‘lipid’ and ‘mRNA’. 100 uL of the prepared lipid formulation wastransferred to tube labeled ‘lipid’.

For the mRNA solution preparation: the amount of mRNA needed for a totalcomplex volume of 200 μL, based on the desired 0.3 mg/mL finalconcentration is 0.6 mg/mL. Based on the concentration of mRNA solution,calculate the necessary volume using the formula (C1)(V1)=(C2)(V2),where C1=stock concentration mRNA, V1=variable, C2=0.6 mg/mL final mRNAconcentration and V2=100 μL of solution volume. Stock mRNA was thawed onice, and mRNA solution was prepared on ice. For experiments, Fireflyluciferase mRNA (Trilink® #L-6107) was purchased at a concentration of 2mg/mL. To achieve the desired concentration, Applicants used 30 μL ofthe stock mRNA solution. To maintain a concentration of 25% EtOH (lipidformulation is at a 25% EtOH) the mRNA is prepared in a solution of 25%EtOH. To the tube labeled mRNA, 30 μL of undiluted mRNA, 25 uL of 100%EtOH and 45 μL of Ultrapure DNase/RNase-Free distilled water(Thermofisher #10977-015) were combined.

Once both tubes were prepared, mRNA tube was added to the lipid tube andmixed well. Tube was briefly vortexed and then heated at 50° C. for 30minutes. Following complexation, complex was transferred to a dialysiscolumn with an 8-10 kD molecular weight cut off (Spectrum labs,Float-a-Lyzer dialysis device #G235031) and dialyzed in 100% PBS (GibcoPBS #10010023) for 2 hours. Following dialysis, sample was transferredto screw-capped plastic sample tube, and either used immediately, orstored at −20° C. until use.

Complex administration in vivo was performed as follows: If using frozencomplex, thaw to room temperature before use, if using freshly dialyzedsamples, proceed immediately. The 200 μL injection volume is accordingto use of a 20 gram mouse. Adjust volume for mice weighing more or lessaccordingly. To perform tail vein injection, restrain animal using arestraining device. A warm water bath or heat lamp can be used to helpdilate and visualize the vein. Disinfect the tail using alcohol. Insertneedle into vein and slowly inject solution into vein, (20-40μL/second). Successful needle insertion and injection will cause bloodto clear from vein. If this does not occur, and resistance is felt uponinjection, remove needle and repeat proximal to primary injection site.Following complete injection of the complex solution, withdraw theneedle and apply gentle compression to the site of injection to stopbleeding.

3 hours following injection of luciferase mRNA, 50 μL of luciferinsubstrate was injected IP into the mouse and allowed to incubate for 15minutes. Immediately, tissues were collected for luciferase activityassay. For experiments, Applicant's utilized an IVIS Lumina LTpre-clinical in vivo imaging system (Perkin-Elmer #IVISLMLT) to measurethe luciferase activity in the individual organs. Using Living Image®software, the flux of signal representing bioluminescence from aspecified region of interest was measured and quantified.

TABLE 1 Volume of Stock solutions (μL) DHDMS HDMS DOPE Cholesterol PEGEtOH 3.10 62.7 568 160.9 26.1 19.2 163 3.14 83.6 582.9 130.8 5.2 38.5159 3.19 60.1 672.6 100.6 52.3 38.5 76 38.53.20 47 568 160.9 52.3 38.5133.3 2.2 146.3 358.7 160.9 73.2 9.6 251.3

TABLE 2 Top producing lipid nanoparticle formulations. PEG PEG CHO-chain Mol DH- LES- Formulation length Weight DMS HDMS DOPE TEROL PEGBruce #3.10 14 5000 0.24 0.38 0.32 0.05 0.01 Bruce #3.14 14 5000 0.32 039 0.26 0.01 0.02 Bruce #3.20 14 5000 0.18 0.38 0.32 0.10 0.02 Bruce#3.19 14 5000 0.23 0.45 0.20 0.10 0.02 Bruce #3.11 14 5000 0.18 0.510.20 0.10 0.01 Bruce #3.15 14 5000 0.27 0.38 0.32 0.01 0.02 Bruce #3.1214 5000 0.25 0.38 0.26 0.10 0.01 Bruce #2.2 18  750 0.28 0.24 0.32 0.140.02 Bruce #3.16 14 5000 0.18 0.47 0.32 0.01 0.02 Bruce #4 14 2000 0.240.40 0.24 0.10 0.02 Bruce #3.18 14 5000 0.32 0.38 0.20 0.08 0.02 MOLAR0.18- 0.24- 0.20- 0.01- 0.01- RANGES 0.32 0.51 0.32 0.14 0.02

TABLE 3 Non-targeting, low performance lipid nanoparticle formulation.PEG PEG CHO- chain Mol DH- LES- Formulation length Weight DMS HDMS DOPETEROL PEG Bruce #23 14 5000 0.10 0.10 0.10 0.38 0.02 Bruce #64 14 50000.10 0.10 0.40 0.30 0.10 Bruce #24 14 5000 0.10 0.10 0.40 0.38 0.02Bruce #52 18  750 0.22 0.10 0.22 0.40 0.06 Bruce #61 14 2000 0.22 0.220.22 0.24 0.10 Bruce #27 14 5000 0.10 0.38 0.10 0.40 0.02 WORKING 0.18-0.24- 0.20- 0.01- 0.01- MOLAR 0.32 0.51 0.32 0.14 0.02 RANGES

Example 4: Novel In Vivo Platform for Lung Delivery

FIG. 19 shows Invivofectamine® Lung (IVF® Lung) research and developmentworkflow.

Reagent comparative analysis was performed using four differentformulations (IVF® Lung, DOTMA:DOPE, DOTAP:DOPE, and Jet PEI®). Animalsreceived systemic delivery of formulations through intravenousinjection. All formulations delivered Trilink® Firefly luciferase mRNAas a payload. FIGS. 20A-20B show experimental results.

Multiple mRNA sequences were tested to determine delivery of mRNA toorgan (i.e., lung) using IVF® Lung. To determine the delivery of FireflyLuciferase, obtained from Trilink® or developed in house,bioluminescence imaging of organ and mouse was used. To determinedelivery of lacZ (obtained from Trilink®), histology on lung tissue wascompleted by staining with beta-gal. To determine delivery of Cre(obtained from Trilink®) in a lacZ Cre-reporter strain,immunofluorescence staining to detect protein expression was used.

mRNA expression kinetics can differ based on optimization of mRNA. FIG.21 shows lung radiance following systemic (intravenous) delivery ofInvivofectamine® Lung including either Trilink® Firefly Luciferase or inhouse optimized Firefly Luciferase.

To assess IVF® Lung delivery of lacZ mRNA and IVF® Lung delivery of CremRNA animals were injected intravenously with LNP complexed. Tissues washarvested 4 hours post-delivery and immediately cryopreserved.Cryosectioning of isolated lung tissue was subsequently stained forbeta-gal and tissue was counterstained for anatomical features (FIGS.24A-24D) or used for immunofluorescence detection of lacZ proteinexpression (FIGS. 25A-25B), depending on the experiment. Delivery ofmRNA was seen demonstrated by positive 3-gal staining andimmunofluorescent staining. Using the reporter strain, specific deliveryto the airways is seen with the fluorescent staining.

FIG. 22 demonstrates the ability of IVF® Lung to not only delivery siRNAto the lung but to endothelial cells as demonstrated by knockdown ofTie2, and endothelial cell specific marker.

FIGS. 27A-27K show that the N/P ratio (see FIG. 26) affectsbiodistribution patterns. However, using the core lipid (DHDMS)formulated with DOPE resulted in no lung delivery and varied N/P ratiodid not affect lung distribution (FIG. 28).

REFERENCES

-   1. Kranz L M, Diken M, Haas H, Kreiter S et al. Systemic RNA    delivery to dendritic cells exploits antiviral defense for cancer    immunotherapy. Nature. 2016 Jun. 1; 534(7607):396-401-   2. Azarmi S, Roa W H, Lobenberg R, Targeted delivery of    nanoparticles for the treatment of lung diseases Adv Drug Deliv Rev,    2008 May 22; 60(8):863-75-   3. Scott McIvor, R. Therapeutic Delivery of mRNA: The Medium Is the    Message. Molecular Therapy 19.5 (2011): 822 823. PMC. Web. 8 Nov.    2015.-   4. Sahin, Ugur, Katalin Karikó, and Özlem Türeci. “mRNA-based    therapeutics [mdash] developing a new class of drugs.” Nature    Reviews Drug Discovery (2014).-   5. Kauffman, Kevin John, et al. “Optimization of Lipid Nanoparticle    Formulations for mRNA Delivery in vivo with Fractional Factorial and    Definitive Screening Designs.” Nano Letters (2015).

1. A composition comprising: (i) a first cationic lipid at acompositional molar ratio from about 0.18 to about 0.32 and of formula:

wherein R¹ and R² are independently substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R³and R⁴ are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; m isan integer from 1 to 6; X_(a) ⁻ is an anion; (ii) a second cationiclipid at a compositional molar ratio from about 0.24 to about 0.51 andof formula:

wherein R⁵ and R⁸ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; R⁶ and R⁷ are independently substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 1 to 6; and X_(b) ⁻ is ananion; (iii) a first helper lipid at a compositional molar ratio fromabout 0.20 to about 0.32; (iv) a second helper lipid at a compositionalmolar ratio from about 0.01 to about 0.14; and (v) a biostabilityenhancing agent at a compositional molar ratio from about 0.01 to about0.02.
 2. The composition of claim 1, wherein m is an integer from about1 to 5, about 1 to 4, about 1 to 3, or where m is 1, 2, 3, 4, 5, or 6.3-10. (canceled)
 11. The composition of claim 1, wherein n is an integerfrom about 1 to 5, about 1 to 4, about 1 to 3, or where n is 1, 2, 3, 4,5, or
 6. 12-19. (canceled)
 20. The composition of claim 1, wherein saidfirst cationic lipid is present at a compositional molar ratio of about0.18 about 0.23, about 0.24, about 0.25, about 0.27, about 0.28, orabout 0.32. 21-26. (canceled)
 27. The composition of claim 1, whereinsaid first cationic lipid has the formula:

wherein X_(a) ⁻ is Cl⁻ or CH₃COO⁻.
 28. The composition of claim 27,wherein X_(a) ⁻ is CH₃COO⁻.
 29. The composition of claim 1, wherein saidfirst cationic lipid is dihydroxy dimyristyl spermidine.
 30. Thecomposition of claim 1, wherein said second cationic lipid is present ata compositional molar ratio of about 0.24, about 0.38 about 0.39 about0.40 about 0.45 about 0.47 about 0.51. 31-36. (canceled)
 37. Thecomposition of claim 1, wherein said second cationic lipid has theformula:

wherein X_(b) ⁻ is Cl⁻ or CH₃COO⁻.
 38. The composition of any one ofclaim 37, wherein X_(b) ⁻ is CH₃COO⁻.
 39. The composition of claim 1,wherein said second cationic lipid is hydroxy dimyristyl spermidine. 40.The composition of claim 1, wherein said first helper lipid is presentat a compositional molar ratio of about 0.20, about 0.24, about 0.26, orabout 0.32. 41-43. (canceled)
 44. The composition of claim 1, whereinsaid first helper lipid is dioleoylphosphatidylethanolamine (DOPE). 45.The composition of claim 1, wherein said second helper lipid is presentat a compositional molar ratio of about 0.01, about 0.05, about 0.08,about 0.10, about 0.14. 46-49. (canceled)
 50. The composition of claim1, wherein said second helper lipid is cholesterol.
 51. The compositionof claim 1, wherein said biostability enhancing agent is present at acompositional molar ratio of about 0.01 or about 0.02.
 52. (canceled)53. The composition of claim 1, wherein said biostability enhancingagent is a polyether compound, a PEGylated phospholipid, or polyethyleneglycol.
 54. (canceled)
 55. (canceled)
 56. The composition of claim 1,wherein said biostability enhancing agent has a molecular weight ofabout 750 g/mol, about 2000 g/mol, or about 5000/mol.
 57. (canceled) 58.(canceled)
 59. The composition of claim 1, wherein said biostabilityenhancing agent is C14 polyethylene glycol 750, C14 polyethylene glycol2000, or C14 polyethylene glycol
 5000. 60-72. (canceled)
 73. Thecomposition of claim 1, further comprising a bioactive agent.
 74. Thecomposition of claim 73, wherein said bioactive agent is a therapeuticagent or a diagnostic agent.
 75. The composition of claim 73, whereinsaid bioactive agent comprises a nucleic acid, a ribonucleoprotein or asmall molecule.
 76. The composition of claim 75, wherein said nucleicacid is an mRNA, a siRNA, a miRNA or a guide RNA.
 77. The composition ofclaim 76, wherein said bioactive agent comprises a nucleic acid and aribonucleoprotein.
 78. The composition of claim 77, wherein saidribonucleoprotein is CRISPR associated protein 9 (Cas9).
 79. Apharmaceutical composition comprising a composition of claim 76 and apharmaceutically acceptable excipient.
 80. A cell comprising acomposition of claim
 76. 81-87. (canceled)
 88. The cell of claim 80,wherein said cell is an epithelial cell, an epithelial lung cell, anendothelial cell, or an endothelial lung cell. 89-92. (canceled)
 93. Thecell of claim 80, wherein said cell is in the lung tissue of a mammal,optionally wherein said mammal is a primate, optionally wherein saidprimate is a human patient.
 94. A method of delivering a bioactive agentto a cell, said method comprising: (i) admixing an bioactive agent witha composition of one of claim 1, thereby forming a bioactive agent-lipidcomplex; (ii) contacting a cell with said bioactive agent-lipid complex,thereby delivering said bioactive agent-lipid complex to said cell. 95.(canceled)
 96. The method of claim 94, wherein said bioactive agent is atherapeutic agent or a diagnostic agent selected from a nucleic acid, aribonucleoprotein a small molecule, or combinations thereof, whereinsaid nucleic acid is an mRNA, a siRNA, miRNA or guide RNA, wherein saidbioactive agent comprises a guide RNA and a ribonucleoprotein, whereinsaid ribonucleoprotein is CRISPR associated protein, optionally whereinsaid CRISPR associated protein is bound to said guide RNA. 97-101.(canceled)
 102. The method of claim 94, wherein said cell is a mammaliancell, optionally wherein said mammalian cell is a primate cell. 103-108.(canceled)
 109. The method of claim 94, wherein said cell is anepithelial cell, an epithelial lung cell, an endothelial, or cell anendothelial lung cell. 110-112. (canceled)
 113. A method of delivering abioactive agent to lung tissue in a subject, said method comprising: (i)admixing an bioactive agent with a composition of claim 1, therebyforming a bioactive agent-lipid complex; (ii) systemically administeringan effective amount of said bioactive agent-lipid complex to a subject,thereby delivering said bioactive agent-lipid complex to a lung tissuein a subject.
 114. (canceled)
 115. A method of treating a pulmonarydisease in a subject in need thereof, said method comprisingadministering to a subject a therapeutically effective amount of abioactive agent and a composition of claim 1, thereby treating saidpulmonary disease in said subject.
 116. The method of claim 113, whereinsaid composition and said bioactive agent are admixed prior to saidadministering.
 117. The method of claim 113, wherein said bioactiveagent comprises a nucleic acid, a ribonucleoprotein or a small molecule.118. The method of claim 117, wherein said nucleic acid is an mRNA, asiRNA, a miRNA or a guide RNA.
 119. The method of one of claim 115,wherein said pulmonary disease is asthma, chronic obstructive pulmonarydisease (COPD), lung cancer or cystic fibrosis.